CN102753187A - Peptide dicer substrate agents and methods for their specific inhibition of gene expression - Google Patents

Peptide dicer substrate agents and methods for their specific inhibition of gene expression Download PDF

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CN102753187A
CN102753187A CN2010800343567A CN201080034356A CN102753187A CN 102753187 A CN102753187 A CN 102753187A CN 2010800343567 A CN2010800343567 A CN 2010800343567A CN 201080034356 A CN201080034356 A CN 201080034356A CN 102753187 A CN102753187 A CN 102753187A
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dsrna
peptide
chain
nucleotide
composition isolated
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苏杰特·库马尔·巴苏
鲍勃·戴尔·布朗
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Dicerna Pharmaceuticals Inc
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Abstract

This invention relates to compounds, compositions, and methods useful for reducing a target RNA and protein levels via use of Dicer substrate siRNA (DsiRN A) -peptide conjugates.

Description

The method of peptide-DICER substrate reagent and specific inhibition of gene expression thereof
The cross reference of related application
The application relates to and the priority of No. the 61/183rd, 818, the U.S. Provisional Patent Application of No. the 61/183rd, 815, U.S. Provisional Patent Application requiring according to 35 U.S.C. § 119 (e) to submit on June 3rd, 2009 and submission on June 3rd, 2009.This paper is incorporated in whole instructions of these applications by reference into.
Technical field
The present invention relates to peptide-dicer substrate conjugate and method for using thereof.
Background of invention
Owing to need safety, delivery of therapeutic molecule effectively, so the fit evaluation of peptide is very important.It is fit (at Beldhoen 2008 Int.J.Mol.Sci 9:1276-1320 to have described peptide; Moschos etc., 2007 Biochemical Society Transaction, the 35th volume, the 4th part: summarize among the 807-810).
The invention summary
The present invention relates to contain the compositions of the double-stranded RNA of puting together with peptide (" dsRNA ") and be used to prepare said method for compositions.DsRNA of the present invention has through puting together double-stranded RNA, siRNA (siRNA) and the Dicer substrate siRNA (" DsiRNA ") of the structure of optimizing with peptide; Be used for effectively sending with targeting and as effectively working with inhibitor efficiently, randomly said dsRNA has the inhibitory action persistent period of prolongation.
The present invention is provided for improving the dsRNA-peptide conjugate of sending with targeting.
In one embodiment; The present invention provides a kind of isolating double stranded RNA (dsRNA) compositions; Said composition comprises and has 5 ' first oligonucleotide chain of end and 3 ' end and second oligonucleotide chain with 5 ' end and 3 ' hold; The length of wherein said first chain and said second chain is at least 16 and 50 nucleotide at the most, and wherein peptide is puted together with said dsRNA and wherein said dsRNA-peptide conjugate combines with target.
In another embodiment; The present invention provides a kind of isolating double stranded RNA (dsRNA) compositions; Said composition comprises and has 5 ' first oligonucleotide chain of end and 3 ' end and second oligonucleotide chain with 5 ' end and 3 ' hold; The length of wherein said first chain and said second chain is at least 16 and 50 nucleotide at the most, wherein said peptide and said dsRNA put together and wherein said dsRNA-peptide conjugate by the target cell internalization.
In one aspect, the length of said first chain and said second chain is at least 25 and 35 nucleotide, at least 19 and 35 nucleotide, at least 19 and 24 nucleotide, at least 25 and 30 nucleotide, at least 26 and 30 nucleotide or at least 21 and 23 nucleotide at the most at the most at the most at the most at the most at the most.
In yet another aspect, second chain comprises outstanding at 3 ' end.
In yet another aspect, first chain comprises outstanding at 3 ' end.
In yet another aspect, at least one in said second chain and said first chain comprises outstanding at 3 ' end.
In yet another aspect, the said 3 ' outstanding nucleotide of said first chain and/or said second chain comprises the nucleotide through modifying.
In yet another aspect, 3 ' outstanding length is 1-5 nucleotide.
In yet another aspect, each the bar chain in said first chain and said second chain is made up of the nucleotide residue of similar number.
In yet another aspect, last residue formation base mismatch of the said 3 ' end of last residue of the said 5 ' end of said first chain and said second chain is right.
In yet another aspect, last residue formation base mismatch of the said 5 ' end of last residue of the said 3 ' end of said first chain and said second chain is right.
In yet another aspect, last residue of the said 3 ' end of last residue of the said 5 ' end of said first chain and penult residue and said second chain and two base mismatch of penult residue formation are right.
In others, it is right that last residue of last residue of the said 3 ' end of said first chain and the said 5 ' end of penult residue and said second chain and penult residue form two base mismatch.
In yet another aspect, peptide comprises 6-100 aminoacid.
In yet another aspect, peptide comprises 10-50 aminoacid.
In yet another aspect, peptide comprises 15-30 aminoacid.
In yet another aspect, peptide comprises 10 aminoacid.
In yet another aspect, dsRNA-peptide conjugate and receptors bind.
In yet another aspect, the dsRNA-peptide conjugate combines with at least one member of ldl receptor family.
In yet another aspect, peptide is the PAR part.
In yet another aspect, peptide is the PAR1 part.
In yet another aspect, peptide is the somatomedin part.
In yet another aspect, peptide is insulin or insulin like growth factor part.
In yet another aspect, peptide is the IGF-1 part.
In yet another aspect, peptide is the hormone part.
In yet another aspect, peptide is the PTH part.
In yet another aspect, peptide is the PTH-1 part.
In yet another aspect, the dsRNA-peptide conjugate combines with receptor binding protein.
In yet another aspect, with stable connexon peptide and said dsRNA are puted together.
In yet another aspect, stable connexon comprises the homotype bi-functional cross-linking agent.
In yet another aspect, stable connexon comprises special-shaped bi-functional cross-linking agent.
In yet another aspect, stable connexon comprises three functional cross-link agents.
In yet another aspect, with the connexon of cleavable peptide and said dsRNA are puted together.
In yet another aspect, the connexon of cleavable comprises the disulphide connexon.
In yet another aspect, with the carbon connexon peptide and said dsRNA are puted together.
In yet another aspect, the carbon connexon comprises 18 carbon at the most.
In yet another aspect, the carbon connexon comprises 6 carbon.
In yet another aspect, under the situation of no connexon, peptide and said dsRNA are puted together.
In yet another aspect, 3 of first chain of peptide and said dsRNA ' end is puted together.
In yet another aspect, 3 of said second chain of peptide and said dsRNA ' end is puted together.
In yet another aspect, 5 of first chain of peptide and said dsRNA ' end is puted together.
In yet another aspect, 5 of said second chain of peptide and said dsRNA ' end is puted together.
In yet another aspect, with 5 of 5 of first chain of peptide and said dsRNA ' end and said second chain ' hold and put together.
In yet another aspect, with 5 of said first chain of peptide and said dsRNA ' end and said second chain said 3 ' hold and put together.
In yet another aspect, with 3 of 3 of first chain of peptide and said dsRNA ' end and said second chain ' hold and put together.
In yet another aspect, with 5 of 3 of first chain of peptide and said dsRNA ' end and said second chain ' hold and put together.
In yet another aspect, the said first chain inside of at least one peptide and said dsRNA is puted together.
In yet another aspect, the said second chain inside of at least one peptide and said dsRNA is puted together.
In yet another aspect, the said first chain inside of at least one peptide and said dsRNA is puted together and the said second chain inside of at least one peptide and said dsRNA is puted together.
In yet another aspect, at least two peptides and said dsRNA are puted together.
In yet another aspect, at least two peptides are identical.
In yet another aspect, at least two peptides are inequality.
In yet another aspect, compositions also comprises at least one dye molecule, and in said dye molecule and said dsRNA and the said peptide at least one puted together.
In yet another aspect, dye molecule is a polyaromatic.
In yet another aspect, dyestuff is a fluorescent dye.
In yet another aspect, compositions also comprises therapeutic agent.
In yet another aspect, therapeutic agent is a cancer therapy drug.
In yet another aspect, cancer therapy drug is selected from paclitaxel, tamoxifen, cisplatin, amycin and vinblastine.
In yet another aspect, therapeutic agent is the medicine that is used to treat metabolic disease or disease.
In yet another aspect, peptide comprises the part of the targeting moiety of toxin.
In yet another aspect, neurotoxin is a clostridial neurotoxins.
In yet another aspect, said compositions also comprises at least a peptide of sending.
In yet another aspect, from first nucleotide (position 1) beginning of 3 of first oligonucleotide chain of said dsRNA ' end, with nucleotide the position of substitution 1,2 and/or 3 through modifying.
In yet another aspect, be deoxyribonucleotide through the nucleotide of modifying.
In yet another aspect, one or two in first oligonucleotide chain and second oligonucleotide chain comprises 5 ' phosphoric acid.
In yet another aspect, at least one nucleotide of said first chain or said second chain is through modifying.
In yet another aspect, through the nucleotide residue of modification be selected from 2 '-O-methyl, 2 '-methoxy ethoxy, 2 '-fluorine, 2 '-pi-allyl, 2 '-O-[2-(methylamino)-2-oxoethyl], 4 '-sulfo-, 4 '-CH2-O-2 '-bridge, 4 '-(CH2) 2-O-2 '-bridge, 2 '-LNA, 2 '-amino and '-O-(N-methyl carbamate).
In yet another aspect, said dsRNA in said cell by the cracking of Dicer endogenous.
In yet another aspect; In said cellular environment, the amount that is enough to reduce the said isolating double-strandednucleic acid of expression of target gene be selected from 1 nanomole or still less, 200 picomoles or still less, 100 picomoles or still less, 50 picomoles or still less, 20 picomoles or still less or still less with 10 picomoles.
In yet another aspect, first chain and second chain link through chemical connexon.
In yet another aspect, 3 of said first chain ' end links through chemical connexon with the said 5 ' end of said second chain.
In yet another aspect, the nucleotide that replaces said second chain or first chain with the directed nucleotide of guiding Dicer cracking through modifying.
In yet another aspect; Composition isolated comprises the nucleotide through modifying; The nucleotide that this process is modified be selected from deoxyribonucleotide, bi-deoxyribose nucleotide, acyclic nucleotide, 3 '-deoxyadenosine (cordycepin), 3 '-azido-3 '-AZT (AZT), 2 '; 3 '-didanosine (ddI), 2 '; 3 '-two deoxidations-3 '-sulfo-cytidine (3TC), 2 '; 3 '-two dehydrogenations-2 ', 3 '-videx (d4T), 3 '-azido-3 '-monophosphic acid nucleotide, 2 of AZT (AZT) ', 3 '-two deoxidations-3 '-sulfo-cytidine (3TC) and 2 '; 3 '-two dehydrogenations-2 ', 3 '-monophosphic acid nucleotide of videx (d4T), 4-thiouracil, 5-bromouracil, 5-iodouracil, 5-(the amino pi-allyl of 3-)-uracil, '-O-alkyl ribonucleotide, 2 '-O-methyl ribonucleotides, 2 '-amino ribonucleotide, 2 '-fluorine ribonucleotide and lock nucleic acid.
In another embodiment, composition isolated comprises the phosphoric acid backbone modification, and this phosphoric acid backbone modification is selected from phosphate ester, thiophosphate and phosphotriester.
In yet another aspect, the nucleotide residue through modifying of the said 3 ' end of said first chain is selected from deoxyribonucleotide, acyclic nucleotide and fluorescence molecule.
In yet another aspect, at least one nucleotide formation base mismatch of at least one nucleotide of said first chain and said second chain is right.
In yet another aspect, send peptide and have the aminoacid sequence that is selected from SEQ ID NO:1-89.
In yet another aspect, compositions is a pharmaceutical composition.
In another embodiment; The present invention provides a kind of method that is used for reducing the expression of target gene of cell; This method comprises: with compare the amount that can effectively reduce the expression of target gene in the cell with reference to dsRNA, cell is contacted with composition isolated of the present invention.
In another embodiment, the present invention provides a kind of optionally cytostatic method that is used for, and this method comprises to be made cell and be enough to cytostatic a certain amount of said composition isolated of the present invention and contact.
In another embodiment; The present invention provides a kind of method that is used for reducing the expression of target gene of animal; This method comprises: with compare the amount that can effectively reduce the expression of target gene in the zooblast with reference to dsRNA, with composition isolated of the present invention treatment animal.
In one aspect, when comparing with suitable contrast dsRNA, composition isolated has enhanced pharmacokinetics.
In yet another aspect, when comparing with suitable contrast dsRNA, dsRNA has enhanced pharmacodynamics.
In yet another aspect, when comparing with suitable contrast dsRNA, dsRNA has the toxicity of reduction.
In yet another aspect, when comparing with suitable contrast dsRNA, dsRNA has picked-up in the enhanced cell.
In another embodiment; The present invention provides a kind of pharmaceutical composition that is used for reducing the expression of target gene of curee's cell, and this pharmaceutical composition comprises and the composition isolated of the present invention of comparing the amount that can effectively reduce the expression of target gene in the cell with reference to dsRNA and pharmaceutically acceptable carrier.
In another embodiment, the present invention provides a kind of method that is used for synthetic dsRNA-peptide conjugate of the present invention, and this method comprises through chemical method or the synthetic said dsRNA of enzyme process.
In another embodiment, the present invention provides a kind of test kit, and said test kit comprises dsRNA-peptide conjugate of the present invention and operation instruction thereof.
The accompanying drawing summary
Fig. 1 (A-H) presents the exemplary configurations according to useful dsRNA-peptide conjugate of the present invention." P "=according to peptide of the present invention (A-flush end-flush end), (B and C-flush end-jag), (the asymmetric end of D and E-) and (F and G-mispairing end).
Fig. 2 illustrates the exemplary sequence of HPRT1 targeting dsRNA of the present invention and KRAS targeting dsRNA.Underlined residue indication 2 '-position that the O-methyl is modified.The expectation site of dicer enzymatic lysis in the arrow indication dsRNA, and dotted line is indicated the cracked estimating position of Argonaute2 mediation in the corresponding target RNA sequence.
Fig. 3 illustrates the exemplary peptides sequence that peptide of the present invention is puted together dsRNA.Also listed by the bonded target of peptide.
Fig. 4 schematic representation exemplary DsiRNA-peptide conjugate of the present invention, and the bulk of molecule of suitably puting together that is shown in swimming lane 2,3,5 and 6 that is numbered 2,3,5 and 6 corresponding DsiRNA-peptide conjugate changes.The dicer enzymatic lysis site of estimating in arrow indication DsiRNA in the sketch map and the DsiRNA-peptide conjugate.
Fig. 5 schematic representation other exemplary DsiRNA-peptide conjugate of the present invention, and indication is numbered being shown in of 2 and 3 DsiRNA-peptide conjugate and suitably puts together the bulk of molecule change in swimming lane 2 and 3.The dicer enzymatic lysis site of estimating in arrow indication DsiRNA in the sketch map and the DsiRNA-peptide conjugate.
Fig. 6 schematic representation other exemplary DsiRNA-peptide conjugate of the present invention, and the bulk of molecule of suitably puting together that is shown in swimming lane 2,3,4 and 5 that is numbered 2,3,4 and 5 corresponding DsiRNA-peptide conjugate changes.The dicer enzymatic lysis site of estimating in arrow indication DsiRNA in the sketch map and the DsiRNA-peptide conjugate.
Fig. 7 schematic representation exemplary DsiRNA-peptide conjugate (comprising cleavable peptide conjugate of the present invention), and the bulk of molecule of suitably puting together that is shown in swimming lane 2,3,4 and 5 that is numbered 2,3,4 and 5 corresponding DsiRNA-peptide conjugate changes.The dicer enzymatic lysis site of estimating in arrow indication DsiRNA in the sketch map and the DsiRNA-peptide conjugate.
Fig. 8 schematic representation exemplary DsiRNA-cyclic peptide conjugate of the present invention, and the bulk of molecule of suitably puting together that is shown in the swimming lane 2 that indication is numbered 2 DsiRNA-peptide conjugate changes.The dicer enzymatic lysis site of estimating in arrow indication DsiRNA in the sketch map and the DsiRNA-peptide conjugate.
Fig. 9 schematic representation exemplary DsiRNA-peptide conjugate of the present invention, the result that the Dicer of DsiRNA shown in it and DsiRNA-peptide conjugate processing is analyzed.
Figure 10 illustrates the block diagram data, and these data show that the DsiRNA-peptide conjugate of transfection is the efficient gene silencing agent that keeps vitro efficacy.In the HeLa cell, carrying out transfection measures.
Figure 11 has showed the serum stability of exemplary DsiRNA-peptide conjugate, and has indicated the half-life.
Figure 12 illustrates the block diagram data, and there is not display body external target gene silencing effect under the vectorial situation of transfection in these tables of data exemplify illustrative DsiRNA-peptide conjugate, and observes sending of raising along with DsiRNA-peptide conjugate concentration constantly increases.In the HeLa cell, measure.
Figure 13 illustrates the block diagram data, and these data are illustrated in and do not have exemplary DsiRNA and the external target gene that strikes in the low HepG2 cell of DsiRNA-peptide conjugate under the vectorial situation of transfection.DsiRNA, DsiRNA-peptide and peptide are that 5 μ M use with concentration.
Figure 14 illustrates IC 50Curve data, these data declarations be exemplary DsiRNA and the external target gene that strikes in the low HepG2 cell of DsiRNA-peptide conjugate under not having the vectorial situation of transfection.Also show the sketch map of test agent.
Detailed Description Of The Invention
The present invention relates to contain the compositions of the double-stranded RNA (" dsRNA ") that comprises peptide; Compare with the dsRNA molecule that does not contain peptide as described herein, said peptide can strengthen sending of dsRNA and/or bio distribution or to the targeting of target and can increase other function and/or strengthen (for example) pharmacokinetics or the pharmacodynamics of this reagent.The invention still further relates to preparation and comprise the method for dsRNA of peptide, the said dsRNA that comprises peptide can be in vivo or the level of external reduction gene and/or expression.
The present invention provides novel dsRNA peptide conjugate.
The present invention also is provided for making the novel dsRNA-peptide conjugate of dsRNA targeting particular organization.Through making the targeting peptide combine to carry out the targeting based on peptide as herein described with surface markers high degree of specificity on destination organization or the tumor.This peptide binding specificity is that dsRNA-peptide conjugate of the present invention provides and makes the ability of dsRNA with the raising of high degree of specificity, selectivity and effective and efficient manner targeting target, and said high degree of specificity, selectivity and effective and efficient manner help dsRNA targeted approach known in the art or reagent.
The present invention provides following advantage.The present invention provides and strengthens the peptide of sending that dsRNA of the present invention sends.The present invention provides near neutrality or is the neutral peptide of sending.For example, nucleic acid and cationic peptide are puted together.TAT (Tat 48-60), wear film peptide (Antp 43-58, oligomerization arginine (R8, R9) etc.) is known in the art.Be different from neutrality of the present invention or approaching neutral peptide, because the polyanion character of nucleic acid, the cationic peptide conjugate is unfavorable for that especially dsRNA puts together.
Peptide of the present invention also is superior to peptide known in the art; Because peptide as herein described need not be connected with dsRNA through the connexon of cleavable but can put together through stable connexon and dsRNA,, the dicer enzyme is suitable for the siRNA molecule in the RISC approach, processed with generation because will processing dsRNA-peptide of the present invention.This is to the pharmaceutical composition advantageous particularly, because stable connexon has improved stability (connexon of cleavable is lost its function thus making and/or the memory period cleavable).
Definition
The present invention is provided for reducing the improved compositions and the method for the expression of target gene in the cell, and the amount that relates to the expression of target gene in effective reduction cell makes target contact with isolating dsRNA.DsRNA molecule of the present invention comprises like defined peptide among this paper so that dsRNA-to be provided peptide conjugate.Compare with the dsRNA reagent that does not contain the corresponding length of passing through the nucleotide pattern of modifying, said peptide strengthens sending of dsRNA and/or bio distribution or is directed against the targeting of target RNA and increases other function, for example pharmacokinetics or pharmacodynamics.
Only if definition in addition, otherwise all scientific and technical terminologies used herein all have the common implication of understanding of person skilled in the art of the present invention.The General Definition of employed many terms among the present invention: Singleton etc. are provided, Dictionary of Microbiology and Molecular Biology (the 2nd edition, 1994) for the technical staff below with reference to document; The Cambridge Dictionary of Science and Technology (Walker compiles, 1988); The Glossary of Genetics, the 5th edition, R.Rieger etc. (volume), Springer Verlag (1991); And Hale&Marham, The Harper Collins Dictionary of Biology (1991).As used herein, except as otherwise noted, otherwise following term has hereinafter to its implication of summing up.
The invention is characterized in one or more dsRNA molecules of puting together with one or more peptides according to the present invention and use these dsRNA molecules to regulate the method for target RNA or coded proteinic level.
DsRNA-peptide of the present invention can and can suppress the expression of target RNA by the dicer enzymatic lysis.
As used herein, " peptide " comprises " sending peptide " and " targeting peptide ".
As used herein, " peptide " is meant linear peptides, branched chain peptide or cyclic peptide.
The invention further relates to and use peptide that dsRNA is transported to required target, for example, on cell or the cell or intracellular receptor, required target tissue or required target cell.
According to the present invention; Required site can be; Such as but not limited to other outside site of brain, adrenal gland or brain (for example, the outer site of cranium), for example; Kidney, liver, pancreas, heart, spleen, the intestines and stomach (GI) road (for example, stomach, intestinal, colon), eye, lung, skin, fat, muscle, lymph node, bone marrow, urinary system and reproductive system (ovary, breast, testis, prostate), Placenta Hominis, hemocyte and combination thereof.Therefore; Required target site can be and is selected from following one or more sites: other site of brain, adrenal gland or brain outside (for example; The outer site of cranium); For example, kidney, liver, pancreas, heart, spleen, the intestines and stomach (GI) road (for example, stomach, intestinal, colon), eye, lung, skin, fat, muscle, lymph node, bone marrow, urinary system and reproductive system (ovary, breast, testis, prostate), Placenta Hominis, hemocyte and combination thereof.
" target cell " is meant like defined any cell among this paper; For example derive from or be present in the cell in any organ; Said organ (for example includes but not limited to other outside site of brain, adrenal gland or brain; The outer site of cranium); For example, kidney, liver, pancreas, heart, spleen, the intestines and stomach (GI) road (for example, stomach, intestinal, colon), eye, lung, skin, fat, muscle, lymph node, bone marrow, urinary system and reproductive system (ovary, breast, testis, prostate), Placenta Hominis, hemocyte and combination thereof.
As used herein, " sending peptide " is meant neutrality or neutral basically peptide." basically neutral " be meant have+5 or the net charge of (for example ,+5 ,+4 ,+3 ,+2 ,+1 or 0) still less.
" net charge " according to the present invention is by confirming according to methods known in the art.For example, as defined net charge among this paper by the net charge of the sum of the net charge of the sum that obtains cationic amino acid (lysine, arginine, histidine) and anionic amino acid (aspartic acid and glutamic acid) come definite.
As used herein, " sending peptide " is meant at least 6 aminoacid, wherein peptide have approximately+5 or the net charge of (for example ,+5 ,+4 ,+3 ,+2 ,+1 or 0) still less.In one aspect, peptide is 6-100 aminoacid (for example, 6,7,8,9,10,15,20,25,30,35,40,45,50,55,60,65,70,75,80,85,90,95 or 100), and peptide has approximately+5 or net charge still less.In another embodiment, peptide is 10-50 aminoacid (for example, 10,15,20,25,30,35,40,45 or 50 aminoacid) and has pact+5 or net charge still less.In another embodiment, peptide is 15-30 aminoacid (for example, 15,16,17,18,19,20,21,22,23,24,25,26,27,28,29 or 30 aminoacid) and has pact+5 or net charge still less.
" sending peptide " according to the present invention is included as at least 6 aminoacid and is neutral peptide.In one aspect, peptide is 6-100 aminoacid (for example, 6,7,8,9,10,15,20,25,30,35,40,45,50,55,60,65,70,75,80,85,90,95 or 100) and does not have net charge.In another embodiment, peptide is that 10-50 aminoacid (for example, 10,11,12,13,14,15,20,25,30,35,40,45 or 50 aminoacid) and peptide do not have net charge.In another embodiment, peptide is 15-30 aminoacid (for example, 15,16,17,18,19,20,21,22,23,24,25,26,27,28,29 or 30 aminoacid) and does not have net charge.
" sending peptide " according to the present invention also refers at least 6 aminoacid and 19 amino acid whose peptides at the most, and wherein peptide has approximately+5 or the net charge of (for example ,+5 ,+4 ,+3 ,+2 ,+1 or 0) still less.
As used herein, " sending peptide " is meant at least 6 aminoacid, wherein peptide have approximately+5 or still less (for example ,+5 ,+4 ,+3 ,+3 ,+2 ,+1 or 0) net charge, and wherein peptide has at least one anionic amino acid.In one aspect, peptide is 6-100 aminoacid (for example, 6,7,8,9,10,15,20,25,30,35,40,45,50,55,60,65,70,75,80,85,90,95 or 100) and has net charge approximately+4 still less.In another embodiment, peptide is 10-50 aminoacid (for example, 10,15,20,25,30,35,40,45 or 50 aminoacid) and has pact+5 or net charge still less.In another embodiment, peptide is 15-30 aminoacid (for example, 15,16,17,18,19,20,21,22,23,24,25,26,27,28,29 or 30 aminoacid) and has pact+5 or net charge still less.
As used herein, " at least one anionic amino acid " is meant at least one in glutamic acid (E) or the aspartic acid (D).For example, XXXEXX or XXXDXX or XXDXEXX or XXXEDXX, wherein X is any aminoacid, wherein peptide have+5 or net charge still less.
The peptide that does not have net charge is meant " neutral peptide ".
As used herein; " neutral peptide " has under neutral pH (for example, pH 6,6.1,6.2,6.3,6.4,6.5,6.6,6.7,6.8,6.9,7,7.1,7.2,7.3,7.4,7.5,7.6,7.7,7.8,7.9,8,8.1,8.2,8.3,8.4 or 8.5) and is approximately 0 net charge.
" neutral peptide " also is included in and has net charge that is approximately 0 and/or the peptide that under about pH 7 (for example, pH 6,6.1,6.2,6.3,6.4,6.5,6.6,6.7,6.8,6.9,7,7.1,7.2,7.3,7.4,7.5,7.6,7.7,7.8,7.9,8,8.1,8.2,8.3,8.4 or 8.5), has isoelectric point, IP (pI) under the neutral pH.
Positively charged aminoacid is lysine (Lys, K), arginine (Arg, R) and histidine (His, H).Electronegative aminoacid is aspartic acid or aspartate (Asp, D), glutamic acid or glutamate (Glu, E).(reference: Lehninger Principles of Biochemistry, the 3rd edition, 2000, compile by David L.Nelson and Michael M., Cox, Worth Publisher, New York, NY).
According to " sending peptide " of the present invention is the aminoacid sequence that when puting together with dsRNA of the present invention, can dsRNA be delivered to suitable target RNA.
" send peptide " and also refer to the aminoacid sequence that when dsRNA and peptide are puted together, can cross over cell membrane transportation dsRNA.
With do not compare with the dsRNA that peptide is puted together, when peptide and dsRNA puted together, useful " sending peptide " improved the internalization of dsRNA to target cell according to the present invention.
With do not compare with the dsRNA that peptide is puted together, when peptide and dsRNA puted together, " sending peptide " useful according to the present invention improved dsRNA sending to target RNA.
As used herein, " raising " is meant that peptide-dsRNA is send 1,2,3,4,5,10,15,20,25,40,35,40,45,50,100,1000 or 10,000 times of the dsRNA that do not put together with peptide or more to sending of target RNA.
As used herein, " raising " is meant that peptide-dsRNA Duos 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% to sending than the sending of dsRNA of not puting together with peptide of target.
By hereinafter " sending " of assessing dsRNA, peptide or dsRNA-peptide conjugate analyzed in described interior fractional analysis or picked-up.
In another embodiment; As used herein " peptide " be meant 6-100 aminoacid (for example, 6,7,8,9,10,15,20,25,30,35,40,45,50,55,60,65,70,75,80,85,90,95 or 100) and with target cell bonded " targeting peptide ".
When with like this paper in definition dsRNA when puting together, " targeting peptide " according to the present invention combines with target or binding site specifically.
As used herein, " combining specifically " is meant hydrogen bonded or the electrostatic attraction with target recipient.
In one aspect, target is receptor or receptor binding protein.
In one aspect, target or binding site or receptor are positioned on the cell surface.
In yet another aspect, target or binding site or receptor are arranged in for example cell (for example, being arranged on Cytoplasm, nucleus or the nucleus surface).
In yet another aspect, target or binding site or receptor are exposed in the solution.
" specificity combination " is through known in the art and measure to confirm (for example, referring to US20080064092 and US2009004174) like defined combination among this paper.In one embodiment, specificity combine be combination and the dsRNA-peptide of sending peptide and described corresponding receptor through dsRNA relatively with other receptor combine confirm that wherein all receptors all exist with mixture.Such as among this paper definition, compare with other receptor, combine with the raising of said receptors bind indication specificity.
In one embodiment, specificity combine be through dsRNA relatively send peptide and said cell combine with dsRNA-peptide and other cell combine confirm that wherein all cells all exists with mixture.As defined among this paper, compare with other cell, combine with the bonded raising indication of said cell specificity.
" specificity combination " is to confirm or definite in vivo with combining of cell through measuring the dsRNA-peptide external through measuring exposed combining of receptor in dsRNA-peptide and the solution.
As used herein, " receptor " comprises the exposed receptor in cell surface receptor, the solution and is positioned at the receptor of cell interior (for example, being arranged on Cytoplasm, nucleus or the nucleus surface).
As used herein, " receptor binding protein " is meant
As used herein " targeting peptide " can carry out at least one in following: when puting together with dsRNA, cross over cell membrane, when puting together with dsRNA according to the present invention, cross over cell membrane transportation dsRNA and the receptor (for example, cell surface receptor) of binding partner when puting together with dsRNA.
In one aspect, " targeting peptide " and the part (for example, the transposition structural domain of neurotoxin) of transposition structural domain or transposition structural domain are puted together.
As used herein, transposition structural domain is meant and helps proteinic penetrating and/or the aminoacid sequence of internalization.
As used herein, the part of transposition structural domain is meant the aminoacid sequence that is enough to keep said function, and said function for example cell guiding gets into or helps cell surface to combine, and for example, combines like defined cell surface receptor among this paper." part of transposition structural domain " also refer to complete amino acid sequence 1% or more, for example 1%, 5%, 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99%.
In one aspect, the targeting peptide can internalization (for example, through directly penetrating or carry out internalization through the endocytosis approach that needs endosome to form, and being also referred to as receptor-mediated endocytosis).
" combination " of dsRNA, peptide or dsRNA-peptide conjugate is to combine to measure through part to assess.
In one embodiment, the binding affinity of the peptide of corresponding receptor or dsRNA-peptide conjugate is about 100 μ M.In another embodiment, the binding affinity of the peptide of corresponding receptor or dsRNA-peptide conjugate is about 1 μ M.In another embodiment, the binding affinity of the peptide of corresponding receptor or dsRNA-peptide conjugate is about 100nM.In another embodiment, the binding affinity of the peptide of corresponding receptor or dsRNA-peptide conjugate is about 10nM.In another embodiment, the binding affinity of the peptide of corresponding receptor or dsRNA-peptide conjugate is about 5nM.In another embodiment, the binding affinity of the peptide of corresponding receptor or dsRNA-peptide conjugate is about 1nM.In another embodiment, the binding affinity of the peptide of corresponding receptor or dsRNA-peptide conjugate is about 0.1nM or littler.(Gauguin etc., J Biol Chem.2008; 283:2604-2613; Grupping etc., Endocrinology 1997; 138 (10): 4064-4068; And Stone, Chervin and Kranz, Immunology.2009; 126 (2): 165-76).
In one embodiment; " targeting peptide " be meant with the bonded 6-100 of a target cell aminoacid (for example; 6,7,8,9,10,15,20,25,30,35,40,45,50,55,60,65,70,75,80,85,90,95 or 100 aminoacid); And at least a portion that comprises the target amino acid sequence, for example, the aminoacid sequence of target peptide.
With do not compare with the dsRNA that peptide is puted together, when peptide and dsRNA puted together, the peptide useful according to the present invention improved the cytotropic targeting of dsRNA.
As used herein, " raising " is meant that peptide-cytotropic targeting of dsRNA conjugate is 1,2,3,4,5,10,15,20,25,40,35,40,45,50,100,1000 or 10,000 times of targeting of the dsRNA that do not put together with peptide or more.
As used herein, " raising " is meant that peptide-cytotropic targeting of dsRNA conjugate Duos 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% than the targeting of the dsRNA that does not put together with peptide.
As used herein; " raising " is meant and do not put together with peptide and compare for amount or the dosage of the required identical dsRNA of the combination, association or the internalization that realize suitable level; Peptide-cytotropic the targeting of dsRNA conjugate (such as hereinafter definition) need still less dsRNA (than the dsRNA of low dosage), this is by the IC in hereinafter described analyzing 50Confirm.For example, as in the body or external measured, with the IC of the identical dsRNA that does not put together with peptide 50Compare, realize that 50% of RNA/ gene expression reduces the IC of required dsRNA-peptide conjugate 50Descend to some extent (for example, referring to Hefner etc., J Biomol Tech.2008 JIUYUE: 19 (4) 231-237; Zimmermann etc., Nature.2006 May 4: 441 (7089): 111-114; Durcan etc., the Mol Pharm.2008 7-8 month; 5 (4): 559-566; Heidel etc., Proc Natl Acad Sci U SA.2007 April 3: 104 (14): 5715-5721).
As used herein, " decline " is meant the IC of dsRNA-peptide conjugate 50IC for the identical dsRNA that do not put together with peptide 501,1/2,1/3,1/4,1/5,1/10,1/15,1/20,1/25,1/40,1/35,1/40,1/45,1/50,1/100,1/1000 or 1/10,000 or littler.
As used herein, " decline " is meant the IC of dsRNA-peptide conjugate 50IC than the identical dsRNA that does not put together with peptide 50Little by 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100%.
In one embodiment, compare with independent dsRNA, the targeting that the dsRNA-peptide conjugate increases is by attachment coefficient K dExpression, it is about 25%.In another embodiment, compare with independent dsRNA, the increase targeting of dsRNA-peptide conjugate is about 100%, that is, compare with independent dsRNA, and the dsRNA-peptide conjugate demonstrates about 1 times increase (that is K of decline, aspect binding affinity d).In another embodiment, compare with independent dsRNA, the dsRNA-peptide conjugate demonstrates about 4 times increase aspect binding affinity.In another embodiment, compare with independent dsiRNA, the dsRNA-peptide conjugate demonstrates about 9 times increase aspect binding affinity.In another embodiment, compare with independent dsRNA, the dsRNA-peptide conjugate demonstrates about 99 times increase aspect binding affinity.In another embodiment, compare with independent dsRNA, the dsRNA-peptide conjugate demonstrates the increase of about 999 times or more times aspect binding affinity.
" combination " is through known in the art and next definite like defined combination mensuration among this paper.In one embodiment, in conjunction be through measure dsRNA send peptide and said receptor combine confirm.
In another embodiment, in conjunction be through measure dsRNA send peptide and said cell combine confirm that wherein all cells all exists with mixture.
" combination " is to confirm or definite in vivo with combining of cell through measuring the dsRNA-peptide external through measuring exposed combining of receptor in dsRNA-peptide and the solution.
As used herein, " targeting " be meant with the receptor mixture in another receptor compare dsRNA peptide conjugate and target recipient preferential or specificity combines or association or internalization.As used herein, " targeting " contain with cell mixture in another receptor on the cell compare the target recipient on dsRNA peptide conjugate and the cell preferential or specificity combines or association or internalization.As used herein, " targeting " contain with cell mixture in another cell compare dsRNA peptide conjugate and cell preferential or specificity combines or association or internalization.That is to say that " targeting " according to the present invention is to confirm in vitro and in vivo or measurement.
" targeting " also refer to " peptide " of the present invention on cell suitable binding site transportation or send, for example, if peptide is a part, targeting is meant peptide sending to the suitable receptor of part, conjugated protein or attachment proteins so.
Can be connected to 5 ' or the 3 ' end of 5 ' or 3 ' end or second chain of first chain of dsRNA of the present invention according to peptide of the present invention; Perhaps be connected to 5 of 5 of the first chain ' end and second chain ' end; Be connected to 3 of 5 of the first chain ' end and second chain ' end, be connected to 5 of 3 of the first chain ' end and second chain ' hold or be connected to, 3 of 3 of the first chain ' end and second chain ' end.
Can also be for example be connected internally to first chain and/or second chain according to peptide of the present invention through the particular functional group on the amino acid residue (for example, Cys last-amino of SH base or Lys).
In one aspect, more than one peptide (for example, dimer, trimer or polymer) is connected to dsRNA.
As used herein; " dimer " is meant two with the directed peptide of puting together each other of any structure (for example through carboxyl and amino terminal linearly or through the covalent bond between the aminoacid of each peptide abreast), and wherein in two peptides also puts together with dsRNA.Dimer also refers to two peptides that put together in the unique site on wherein each peptide and dsRNA.
As used herein, " trimer " is meant three peptides of puting together each other, and wherein in three peptides puts together with dsRNA.Trimer also refers to three peptides that put together in the unique site on wherein each peptide and dsRNA.Trimer refer to also wherein that two peptides in three peptides are puted together each other and wherein a peptide in two peptides also put together with dsRNA and the 3rd peptide and dsRNA on three peptides puting together of unique site.
As used herein, " polymer " is meant more than 1 peptide for example 2,3,4,5,6,7,8,9,10 or more a plurality of.The present invention provides the dsRNA that puts together with a plurality of peptides, and wherein peptide has identical or different sequences.In one embodiment, a plurality of peptides are meant one or more peptide and one or more targeting peptides sent.
Term " peptide " comprises the continuous amino acid of limited quantity, and said aminoacid is the peptide that combines; And said term " peptide " comprises like defined targeting peptide among this paper or sends peptide; No matter said peptide be natural molecule or synthetic (promptly; Natural molecule; Or its variant of modifying through chemical/physical), said targeting peptide or send peptide and can send dsRNA and/or combine with peptide target (for example, the receptor on cell or the cell).
As used herein " peptide " can be derived from natural protein.
As used herein " peptide " can comprise different protein domain (for example, chimeric peptide).
As used herein " peptide " can be based on the structure-function relationship of specific amino acids sequence and the synthetic peptide that designs, and needn't have homology with native sequences.
Peptide of the present invention and dsRNA of the present invention put together.
As used herein, put together and be meant through any covalently or non-covalently association known in the art and connect.
Can peptide of the present invention and dsRNA of the present invention be puted together through any amino acid residue in the peptide; For example; The C terminal amino acid of carboxyl through the C terminal amino acid and C end is puted together or the alpha-amido through the N terminal amino acid and the N terminal amino acid of N end put together perhaps with amino acid residue on particular functional group's (for example, Cys last-amino of SH base or Lys) put together.
Can peptide of the present invention and dsRNA of the present invention be puted together through any amino acid residue in the peptide sequence amino of the intermediary lysine residue of peptide sequence (for example, through).
Can be through stable covalent bond (including but not limited to distance of zero mark degree connexon, the difunctional connexon of homotype, special-shaped difunctional connexon or three function connexons); To put together (reference: Bioconjugate Techniques according to peptide of the present invention and dsRNA of the present invention; 1996, Greg T.Hermanson, Academic Press; San Diego, CA.; Chemistry of Protein Conjugation and Cross-linking, 1991, Shan S.Wong, CRC Press, Boca Raton, FL).
As used herein; " distance of zero mark degree connexon " is meant through reaction and puts together; Wherein under the situation of no connexon, make reactant (for example; Reactive group that dsRNA is last and the functional group on the peptide, the for example free amino group of the active group on the amino acid side chain, terminal amino acid residue and free carboxyl group etc.) condensation puts together molecule with formation.For example, through with the end reactant of peptide and the end reactant reaction of dsRNA, form " distance of zero mark degree connexon ".The instance that the distance of zero mark degree connects includes but not limited to disulphide, amide, ester, thioesters etc.
As used herein, " the difunctional connexon of homotype " is meant and the puting together of connexon with two similar functional groups.That the instance of the difunctional connexon of homotype includes but not limited to is amino directed, that carboxyl is directed, sulfydryl is directed etc.
As used herein, " special-shaped difunctional connexon " be meant with have two not the connexon of homospecific different functional groups put together.The instance of special-shaped difunctional connexon includes but not limited to that amino and sulfydryl are directed, that amino and carboxyl are directed, the combination of carboxyl and sulfydryl orientation etc.
As used herein, " three function connexons " is meant and the puting together of connexon with three reactive functionality.The instance of three function connexons includes but not limited to 4-azido-2-nitrobenzophenone biotin complex of yeast .-4-nitro phenyl ester (ABNP), sulphonyl succinimido-2-[6-(biotin amine)-2-(the p-azido benzoyl is amino) hexanamido] ethyl-1; 3 '-dithio propionic ester (sulfo-SBED), other is based on molecule of biotin complex of yeast. etc.
Can also make according to peptide of the present invention and dsRNA and put together through the connexon (including but not limited to disulphide, ester, ethylene glycol, diazonium and sulfone connexon) of cleavable.
Make according to peptide of the present invention and can put together through the carbon connexon with dsRNA; Said carbon connexon is the carbon connexon of for example one or more carbon (for example, 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25 or more a plurality of carbon).
Can use prothetic group to make according to peptide of the present invention and dsRNA puts together.Prothetic group includes but not limited to metal ion, porphyryl, coenzyme and other non-peptide base section, for example, and carbohydrate or oligosaccharide (Wong, S.S. (1991), Chemistry of protein conjugation and cross-linking, CRC Press).
In one embodiment, through being expressed as fusion constructs peptide and dsRNA are puted together.
Can " peptide " be connected to dsRNA through any conventional chemical conjugation techniques that is well known to those skilled in the art.In this, with reference to Hermanson, G.T. (1996), Bioconjugate techniques, Academic Press and, S.S. (1991), Chemistry of protein conjugation and cross-linking, CRC Press with reference to Wong.
" peptide " puted together with dsRNA is non-covalent.
As used herein, " peptide-dsRNA conjugate " is meant through the peptide that (includes but not limited to connection/conjugation methods as herein described) someway and dsRNA puts together.
In one aspect, peptide-dsRNA conjugate also comprises one or more dye molecules.
As used herein; " dye molecule " includes but not limited to polyaromatic dyestuff or fluorescent dye; For example Cy3, Cy5, Cy5.5, Alexa
Figure BDA0000133674250000211
(for example, Alexa Fluor 488, Alexa Fluor 555, Alexa Fluor 647 etc.).
In one aspect, such as among this paper definition, peptide-dsRNA conjugate also comprises sends peptide.
In one aspect, peptide-dsRNA conjugate also comprises therapeutic agent, for example the reagent of anticarcinogen or treatment metabolic disease or disease.Anticarcinogen includes but not limited to antiviral agent (Fiume etc., FEBS Lett.1983; 153 (1): 6-10), cisplatin (Mukhopadhyay S etc., Bioconjug Chem.2008; 19 (1): 39-49), amycin (Guan H etc., Bioconjug Chem.2008; 19 (9): 1813-21), paclitaxel (Dubikovskaya EA etc., Proc Natl Acad Sci U S A.2008; 105 (34): 12128-33, R é gina A etc., Br J Pharmacol.2008; 155 (2): 185-97), tamoxifen (Rickert etc., Biomacromolecules.2007; 8 (11): 3608-3612) and vinblastine (DeFeo-Jones D etc., Mol Cancer Ther.2002; 1 (7): 451-459).The compositions that comprises the therapeutic agent that makes up with peptide-dsRNA conjugate can comprise 1: 10 respectively; 000 to 1: 1 to 10; 000: 1 reagent/conjugate ratio; For example 1: 5000 to 5000: 1,1: 1000 to 1000: 1,1: 100 to 100: 1,1: 10 to 10: 1, based on molecular weight, mole or actual weight.
" peptide-dsRNA conjugate " is meant that wherein said peptide and said dsRNA keep the molecule of their functions.
As used herein; " decline " (for example; Onset time of dsRNA-peptide conjugate reduces or the delivery rate of dsRNA-peptide conjugate descends) onset time or the delivery rate that are meant the dsRNA-peptide conjugate be 1/1,1/2,1/3,1/4,1/5,1/10,1/15,1/20,1/25,1/40,1/35,1/40,1/45,1/50,1/100,1/1000 or 1/10,000 or lower of the onset time of the identical dsRNA that do not put together with peptide or delivery rate.
As used herein; Onset time or the delivery rate that " decline " (for example, the onset time of dsRNA-peptide conjugate reduces or the delivery rate of dsRNA-peptide conjugate descends) is meant the dsRNA-peptide conjugate reduces by 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% than onset time or the delivery rate of the identical dsRNA that does not put together with peptide.
As used herein, " onset time " is meant that (for example, to human or animal (for example, mice or rat) curee) uses the time period between dsRNA and the dsRNA arrival target RNA in external (for example, to cell or to tissue culture medium (TCM)) or the body.
As used herein, " delivery rate " is meant that dsRNA arrives the required time of target RNA after using dsRNA.
As used herein, " acting duration " is meant that dsRNA suppresses the time period of target rna expression.
As used herein, " contrast " or " reference " (for example, contrast dsRNA) be meant on length with to the special dsRNA of particular target RNA (test dsRNA) quite but to the non-specific dsRNA of particular target RNA.The contrast RNA have with to the special dsRNA of target target nucleotide sequence inequality.Contrast (for example; Control peptide) be meant one or more and suitable in length and the electric charge with peptide (test peptides) that the special dsRNA of target RNA is puted together, but have with and the aminoacid sequence different amino acid sequence of peptide (test peptides) that the special dsRNA of target RNA is puted together.Contrast (for example, contrast dsRNA-peptide conjugate) be meant wherein dsRNA on length with suitable, but to the dsRNA-peptide conjugate of the non-specific dsRNA of particular target RNA to the special dsRNA of particular target RNA.Contrast dsRNA-peptide conjugate also refer on wherein said peptide one or more in length and electric charge with and peptide that the special dsRNA of target RNA is puted together quite but have and and the dsRNA-peptide conjugate of the aminoacid sequence different amino acid sequence of peptide that the special dsRNA of target RNA is puted together.Contrast dsRNA-peptide conjugate also refer on wherein said peptide one or more in length and electric charge with and peptide that the special dsRNA of target RNA is puted together quite but have with and the aminoacid sequence different amino acid sequence of peptide that the special dsRNA of target RNA is puted together, and wherein dsRNA on length with suitable but to the dsRNA-peptide conjugate of the non-specific dsRNA of particular target RNA to the special dsRNA of particular target RNA.
As used herein, test peptides or test dsRNA are meant and comprise peptide or the dsRNA of reduction according to the conjugate of target rna expression of the present invention.Test dsRNA is meant the dsRNA of reduction according to the expression of target RNA of the present invention." test " dsRNA-peptide conjugate comprises the test dsRNA that puts together with test peptides.
As used herein, term " nucleic acid " is meant deoxyribonucleotide, ribonucleotide or nucleotide and the polymer thereof through modifying that is strand or double chain form.This term is contained and is contained known nucleotide analog or the framework residue of process modification or the nucleic acid of binding; These nucleic acid are synthetic, natural in non-natural; Have with reference to the similar binding characteristic of nucleic acid, and according to mode metabolism like ucleotides.The instance of these analog includes but not limited to thiophosphate, phosphoramidate, methyl phosphorodithioate, chirality methyl phosphate ester, 2-O-methyl ribonucleotides, peptide nucleotide (PNA).
As confessed in this area, as used herein " nucleotide " is used to comprise those nucleotide of the base with natural base well known in the art (standard) and process modification.These bases are usually located at 1 ' position of nucleotide sugar part.Nucleotide comprises base, sugar and phosphate usually.Nucleotide can be at sugar, phosphoric acid and/or base portion place without modifying or through modifying, (also replacedly being called nucleotide analog, the nucleotide through modifying, non-natural nucleotides, non-standard nucleotide etc.; For example referring to Usman and McSwiggen, again; Eckstein etc., International PCT discloses WO No. 92/07065; Usman etc., International PCT discloses WO No. 93/15187; Uhlman&Peyman, again is all incorporated this paper at this by reference).There are several instances through the nucleic acid base modified known in the art, equal Nucleic Acids Res.22:2183, summarize in 1994 like Limbach.Some limiting examples that can be introduced into the base modification of nucleic acid molecules comprise: hypoxanthine, purine, pyridine-4-ketone, pyridin-2-ones, phenyl, pseudouracil, 2; 4; 6-trimethoxy-benzene, 3-methyluracil, dihydrouridine, naphthyl, aminophenyl, 5-alkyl cytidine (for example, 5-methylcytidine), 5-alkyl uridnine (for example, thymidine), 5-halo uridnine are (for example; The 5-broxuridine) or 6-aza-pyrimidine or 6-alkyl pyrimidine (for example; The 6-methyluridine), (Burgin etc., Biochemistry 35:14090,1996 such as propine; Uhlman and Peyman, again).In this respect, " through the base of modifying " is meant and is positioned at nucleotide base or its equivalent of 1 ' position except that adenine, guanine, cytosine and uracil.
As used herein, " through the nucleotide of modifying " is meant the nucleotide that nucleoside, nucleoside base, pentose ring or phosphate are carried out one or more modifications.For example, do not comprise ribonucleotide that contains single adenosine phosphate, Guanosine 5'-Monophosphate, single phosphoric acid uridine and single cytidine phosphate and the deoxyribonucleotide that contains dAMP, single phosphate deoxidating deoxyguanosine, single phosphoric acid AZT and single deoxycytidine monophosphate through the nucleotide of modifying.Modification comprises by the enzyme of modified nucleotide (such as, transmethylase) modifies the natural modifications that produces.Nucleotide through modifying also comprises synthetic nucleotide or non-natural nucleotide.Synthetic modification or non-natural modification comprise having 2 ' those modifications of modifying or those modifications that comprise base analogue in the nucleotide, said 2 ' for example modify 2 '-O-methyl, 2 '-methoxy ethoxy, 2 '-fluorine, 2 '-pi-allyl, 2 '-O-[2-(first ammonia is amino)-2-oxoethyl], 4 '-sulfo-, 4 '-CH 2-O-2 '-bridge, 4 '-(CH 2) 2-O-2 '-bridge, 2 '-LNA and '-O-(N-methyl carbamate).About the disclosure described with 2 ' modified nucleotide, " amino " be meant can through modify or without modify 2 '-NH 2Or 2 '-O-NH 2This type for example is described in through the group of modifying in No. the 6th, 248,878, the United States Patent (USP)s such as No. the 5th, 672,695, United States Patent (USP) such as Eckstein and Matulic-Adamic.
About nucleic acid molecules of the present disclosure, modification can be present in according to the pattern of these reagent on the chain or two chains of dsRNA.As used herein; " alternate position " is meant wherein whenever to have one without the nucleotide of modification (for example to exist between through the nucleotide of modifying through on the nucleotide modified or the dsRNA chain of confirming length each at a distance from a nucleotide; Without the ribonucleotide of modifying) pattern (for example, 5 '-MNMNMN-3 '; 3 '-MNMNMN-5 '; Wherein M is without the nucleotide of modifying for the nucleotide N through modification).In certain embodiments; According to any Position Number regulation as herein described; The modification pattern from be positioned at 5 ' or first nucleotide of 3 ' end begin (the terminal residue assigned address 1 of chain after the Dicer cracking incident of estimating with reference to DsiRNA reagent of the present invention in certain embodiments; Therefore, position 1 does not always constitute 3 of preprocessing reagent of the present invention ' end or 5 ' end residue).In other embodiments, reference and 5 of opposite strand ' or the nucleotide residue assigned address 1 of 3 ' end complementary first or second chain.For example, in certain embodiments, position 1 is the nucleotide residue with complementary second chain of 5 ' terminal nucleotide residue of first oligonucleotide chain.The present invention contain the pattern of wherein modifying from 5 ' or 3 ' end (according to any Position Number regulation as herein described) start from the dsRNA of any one position position 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23 or 24.The present invention also contain the pattern of wherein modifying from 5 ' or 3 ' end residue start from dsRNA for any position of at least one nucleotide.
The pattern of passing through the nucleotide of modifying that is in alternate position can run through the total length of chain; But in certain embodiments, said pattern comprises respectively and contains at least 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24 or more a plurality of at least 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28 or more a plurality of nucleotide through the nucleotide modified.
As used herein, " alternate position to " be meant wherein on the dsRNA chain of confirming length two successive through the nucleotide modification by two successive without the separated pattern of modified nucleotide (for example, 5 '-MMNNMMNNMMNN-3 '; 3 '-MMNNMMNNMMNN-5 '; Wherein M is without the nucleotide of modifying for nucleotide and N through modification).In one embodiment, according to any Position Number regulation as herein described, the modification pattern is since first nucleotide position of 5 ' end or 3 ' end.The pattern of passing through the nucleotide of modifying that is in alternate position possibly run through the total length of chain; But preferably, said pattern comprises at least 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28 nucleotide of the nucleotide that contains at least 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24 process modifications respectively.What should stress is, above-mentioned modification pattern is exemplary and do not plan to limit scope of the present invention.
As used herein, " base analogue " is meant the heterocyclic moiety that is arranged in the position 1 through the nucleotide sugar part of the nucleotide modified that can incorporate nucleic acid duplex into ' (maybe can incorporate the equivalent position that the nucleotide sugar of nucleic acid duplex partly replaces into).In dsRNA of the present invention, base analogue is generally purine or pyrimidine bases, does not comprise common base guanine (G), cytosine (C), adenine (A), thymus pyrimidine (T) and uracil (U).Base analogue can form duplex with other base or the base analogue among the dsRNA.Base analogue comprises the base analogue that can be used for Compounds and methods for of the present invention; For example at the United States Patent (USP) the 5th of Benner; 432, No. 272 and the 6th, 001; No. 983 the United States Patent (USP) with Manoharan discloses disclosed base analogue in No. 20080213891, and it incorporates this paper by reference into.The limiting examples of base comprises: hypoxanthine (I), xanthine (X), 3 β-D-ribofuranosyl-(2; The 6-di-amino-pyrimidine) (K), 3-β-D-ribofuranosyl-(1-methyl-pyrazolo [4; 3-d] pyrimidine-5; 7 (4H; 6H)-diketone) (P), iso-cytosine (iso-C), isoguanine (iso-G), 1-β-D-ribofuranosyl-(5-nitroindoline), 1-β-D-ribofuranosyl-(3-nitro-pyrrole), 5-bromouracil, 2-aminopurine, 4-sulfo--dT, 7-(2-thienyl)-imidazo [4; 5-b] pyridine (Ds) and pyrrole-2-aldehyde (Pa), 2-amino-6-(2-thienyl) purine (S), 2-oxo pyridine (Y), difluoro toluene base, 4-fluoro-6-tolimidazole, 4-tolimidazole, 3-methyl isoquinolone base, 5-methyl isoquinolone base and 3-methyl-7-propinyl isoquinolone base, 7-azaindolyl, 6-methyl-7-azaindolyl, imidazopyridyl, 9-methyl-imidazopyridyl, Pyrrolopyrazine base, isoquinolone base, 7-propinyl isoquinolone base, propinyl-7-azaindolyl, 2; 4; 5-trimethylphenyl, 4-methylindole base, 4; 6-dimethylated indolyl, phenyl, naphthyl, anthryl, phenanthryl, pyrenyl, stilbene radicals, naphthacenyl, pentacenyl and structural derivative thereof (Schweitzer etc., J.Org.Chem., 59:7238-7242 (1994); Bergerr etc., Nucleic Acids Research, 28 (15): 2911-2914 (2000); Moran etc., J.Am.Chem.Soc., 119:2056-2057 (1997); Morales etc., J.Am.Chem.Soc., 121:2323-2324 (1999); Guckian etc., J.Am.Chem.Soc., 118:8182-8183 (1996); Morales etc., J.Am.Chem.Soc., 122 (6): 1001-1007 (2000); McMinn etc., J.Am.Chem.Soc., 121:11585-11586 (1999); Guckian etc., J.Org.Chem., 63:9652-9656 (1998); Moran etc., Proc.Natl.Acad.Sci., 94:10506-10511 (1997); Das etc., J.Chem.Soc., Perkin Trans., 1:197-206 (2002) Shibata etc., J.Chem.Soc., Perkin Trans., 1:1605-1611 (2001); Wu etc., J.Am.Chem.Soc., 122 (32): 7621-7632 (2000); O ' Neill etc., J.Org.Chem., 67:5869-5875 (2002); Chaudhuri etc., J.Am.Chem.Soc., 117:10434-10442 (1995); And No. the 6th, 218,108, United States Patent (USP)).Base analogue also can be universal base.
As used herein; " universal base " is meant the position 1 that is arranged in through the nucleotide sugar part of the nucleotide modified ' or be arranged in the heterocyclic moiety of the equivalent position that nucleotide sugar partly replaces; In the time of in being present in nucleic acid duplex; Said heterocyclic moiety can be under the situation that does not change double-spiral structure (for example, the structure of phosphoric acid skeleton) and more than one base relative localizations.In addition, universal base can not destroyed the ability of its single-chain nucleic acid that is positioned at and target nucleic acid formation duplex.Can conspicuous by one of skill in the art method (for example, ultraviolet absorptivity method, circular dichroism spectrometry, gel shift method, single-chain nucleic acid enzyme sensitivity method etc.) mensuration contain the single-chain nucleic acid of universal base and the ability that target nucleic acid forms duplex.In addition, can change and to observe condition (for example, temperature) stable or formation that duplex forms, because melting temperature (Tm) is relevant with the stability of nucleic acid duplex with definite duplex.Compared to accurate complementary with reference to single-chain nucleic acid with target nucleic acid, the Tm that contains the single-chain nucleic acid of universal base and the duplex that target nucleic acid forms is lower than the Tm of the duplex that forms with complementary nucleic acid.Yet, being produced comparing of single mispairing with universal base wherein by base substitution with reference to single-chain nucleic acid, the Tm that contains the single-chain nucleic acid of universal base and the duplex that target nucleic acid forms is higher than the Tm of the duplex that forms with the nucleic acid with base mismatch.
Some universal base can through under the base pair formation condition universal base and all between base guanine (G), cytosine (C), adenine (A), thymus pyrimidine (T) and the uracil (U) the formation hydrogen bond carry out base pairing.Universal base is not the base that only forms base pair with single complementary base.In duplex, universal base can with G, C, A, T and the U relative on the relative chain of duplex with said universal base in each do not form hydrogen bond, form a hydrogen bond or more than a hydrogen bond.Preferably, universal base can not interact with the base relative with said universal base on the relative chain of duplex.In duplex, between universal base, carry out base pairing and do not change the double-spiral structure of phosphoric acid skeleton.Universal base also can interact through the base in the adjacent nucleotide on accumulative facies mutual effect and the identical nucleic acid chain.This accumulative facies mutual effect makes duplex stable, particularly universal base not can with the relative chain of duplex on be positioned at the base relative and form under the situation of any hydrogen bond with said universal base.The limiting examples of general binding nucleotide comprises: (U.S. Patent application of Quay etc. discloses No. 20070254362 for inosine, 1-β-D-ribofuranosyl-5-nitroindoline and/or 1-β-D-ribofuranosyl-3-nitro-pyrrole; Van Aerschot etc., An acyclic 5-nitroindazole nucleoside analogue as ambiguous nucleoside.Nucleic Acids Res.1995 November 11; 23 (21): 4363-70; Loakes etc., 3-Nitropyrrole and 5-nitroindole as universal bases in primers for DNA sequencing and PCR.Nucleic Acids Res.1995 July 11; 23 (13): 2361-6; Loakes and Brown, 5-Nitroindole as an universal base analogue.Nucleic Acids Res.1994 October 11; 22 (20): 4039-43).
As used herein; " ring " is meant the structure by the single chain formation of nucleic acid, and wherein hybridize with the mode that the strand nucleotide district between the complementation district can not carry out duplex formation or Watson-Crick base pairing in lateral complementary district in specific strand nucleotide district.Ring is the strand nucleotide district of any length.The instance of ring comprises the unpaired nucleotide that is present in such as in the structures such as hairpin structure, stem ring or extended loop.
As used herein, " extended loop " is meant single-stranded loop and is positioned at ring lateral other 1,2,3,4,5,6 or reaches 20 base pairs or duplex under the situation of dsRNA.In extended loop, the lateral nucleotide of ring that is positioned on 5 ' side forms duplex with the lateral nucleotide of ring that is positioned on 3 ' side.Extended loop can form hair clip or stem ring.
As used herein, " tetranucleotide ring " is meant the ring of being made up of four nucleotide (strand district) under the situation of dsRNA, and said ring forms stable secondary structure, and said secondary structure helps the stability of the Watson-Crick hybridization nucleotide of adjacency.Under the situation of not accepting the opinion constraint, the tetranucleotide ring can make the Watson-Crick base pair of adjacency stable through the accumulative facies mutual effect.In addition, the interaction between four nucleotide in the tetranucleotide ring includes but not limited to non-Watson-Crick base pairing, accumulative facies mutual effect, hydrogen bond and contact interaction (Cheong etc., 16 days Augusts nineteen ninety of Nature; 346 (6285): 680-2; Heus and Pardi, Science on July 12nd, 1991; 253 (5016): 191-4).The tetranucleotide ring raises the melting temperature (Tm) in abutting connection with duplex, and it is higher than according to by four desired melting temperatures of naive model ring sequence (Tm) of base composition at random.For example, the tetranucleotide ring can be given and comprise the hairpin structure of duplex that length is at least 2 base pairs at 10mM NaHPO 4In at least 55 ℃ melting temperature.The tetranucleotide ring can contain ribonucleotide, deoxyribonucleotide, nucleotide and combination thereof through modifying.The instance of RNA tetranucleotide ring comprises: UNCG family tetranucleotide ring (for example, UUCG), GNRA family tetranucleotide ring (for example, GAAA) with CUUG tetranucleotide ring.(Woese etc., Proc Natl Acad Sci U S are year November A.1990; 87 (21): 8467-71; Antao etc., Nucleic Acids Res.1990 November 11; 19 (21): 5901-5).The instance of DNA tetranucleotide ring (for example comprises d (GNNA) family tetranucleotide ring; D (GTTA)), the tetranucleotide ring (for example, d (TTCG)) of the tetranucleotide ring of d (GNNA) family tetranucleotide ring, d (GNAB) family tetranucleotide ring, d (CNNG) family, d (TNCG) family.(Nakano etc., Biochemistry, 41 (48), 14281-14292,2002; SHINJI etc., Nippon Kagakkai Koen Yokoshu, the 78th volume; The 2nd phase; The 731st page (2000)).
DsRNA compositions of the present invention is because they are through moulding as the substrate of Dicer enzyme entering RNAi path, at least in part owing to the chain length of this based composition, so be also referred to as Dicer substrate siRNA (" DsiRNA ") reagent among this paper." DsiRNA reagent " of the present invention compositions comprises the dsRNA as the precursor molecule that is used for the processing of Dicer enzyme,, processes DsiRNA of the present invention in vivo to produce active siRNA that is.Specifically, DsiRNA is processed into the active siRNA that incorporates among the RISC through Dicer.This precursor molecule that mainly is called " DsiRNA reagent " or " DsiRNA molecule " in this article can also be called as precursor RNA i molecule in this article.As used herein, term " active siRNA " refers to double-strandednucleic acid, and wherein each bar chain comprises RNA, RNA analog or RNA and DNA.SiRNA comprises 19 to 23 nucleotide or comprises 21 nucleotide.Active siRNA has 2bp usually on 3 of each bar chain ' end outstanding, so that the double-stranded tagma among the siRNA comprises 17 to 21 nucleotide, or 19 nucleotide.
In certain embodiments; DsRNA of the present invention includes but not limited to comprise the dsRNA of first chain and second chain, and said first chain and said second chain comprise 16 to 50,19 to 35,19 to 24,25 to 30,25 to 35,26 to 30,21 to 23 nucleotide on length.
DsiRNA reagent of the present invention has sufficient length and produces siRNA so that DsiRNA reagent is processed through Dicer.Therefore, suitable DsiRNA reagent contains an oligonucleotide sequence (first sequence), and the length of said oligonucleotide sequence is at least 25 nucleotide and be no more than about 35 nucleotide.The length of this sequence of RNA can be about 26 to 35,26 to 34,26 to 33,26 to 32,26 to 31,26 to 30 and 26 to 29 nucleotide.The length of this sequence can or be 27 nucleotide for about 27 or 28 nucleotide.Second sequence of DsiRNA reagent can be (for example, in eukaryotic Cytoplasm) and the annealed any sequence of first sequence under biotic factor.Usually; Second oligonucleotide sequence will have at least 19 and the complementary base pair of first oligonucleotide sequence; More generally, second oligonucleotide sequence will have about 21 or more a plurality of or about 25 or the more a plurality of and complementary base pair of first oligonucleotide sequence.In one embodiment, second sequence is identical with first sequence length, and DsiRNA reagent is flush end.In another embodiment, the end of DsiRNA reagent has one or more outstanding.In certain embodiments, wherein second sequence is identical with first sequence length, and it is right that last residue of last residue of the said 3 ' end of said first chain and the said 5 ' end of said second chain forms base mismatch.In other embodiments, wherein second sequence is identical with first sequence length, and it is right that last residue of 3 of last residue of 5 of said first chain ' end and second chain ' end forms base mismatch.In other embodiments, wherein second sequence is identical with first sequence length, and it is right that last residue of 5 of last residue of 3 of first chain ' end and penult residue and second chain ' end and penult residue form two base mismatch.In other embodiments, wherein second sequence is identical with first sequence length, and it is right that last residue of 3 of last residue of 5 of first chain ' end and penult residue and second chain ' end and penult residue form two base mismatch.
In certain embodiments, first oligonucleotide sequence of DsiRNA reagent and second oligonucleotide sequence are present on the independent oligonucleotide chain, and said independent oligonucleotide chain can be and normally chemosynthesis.In some embodiments, the length of two chains is 26 to 35 nucleotide.In other embodiments, the length of two chains is 25 to 30 or 26 to 30 nucleotide.In one embodiment, the length of two chains is 27 nucleotide, and is fully complementary and have a flush end.In one embodiment, one or two oligonucleotide chains can be as the substrates of Dicer.In other embodiments, have at least a modification, it promotes Dicer to be incorporated into the double-stranded RNA structure according to the maximum orientation of the effect that makes the gene expression of double-stranded RNA OILS STRUCTURE DEPRESSION.In certain embodiments of the invention, DsiRNA reagent is made up of the oligonucleotide chain of two different lengths, wherein DsiRNA have flush end at 3 ' end place of first chain (sense strand) and have 3 at 3 ' end place of second chain (antisense strand) ' outstanding.DsiRNA can also contain one or more DNAs (DNA) base and replace.
The suitable DsiRNA compositions that contains two independent oligonucleotide can connect with the outside of chemical mode in their annealed zone through cytotoxic compounds.Known in the art and can use many suitable cytotoxic compounds.Suitable group will can not be blocked Dicer to the activity of DsiRNA and the orientation destruction that can not disturb the RNA that is transcribed by target gene.Perhaps, two independent oligonucleotide can connect through the 3rd oligonucleotide, so that after two oligonucleotide annealing forming the DsiRNA compositions, produce hairpin structure.Hairpin structure will can not be blocked Dicer to the activity of DsiRNA and the orientation destruction that will can not disturb target RNA.
As used herein; Have with the dsRNA of the sequence of target RNA or cDNA sequence " fully complementary " (for example; DsiRNA or siRNA) be meant to have and (for example be enough to through RNAi mechanism; The RISC complex) or the dsRNA of the destructive sequence of target RNA (wherein narrated the cDNA sequence, the RNA sequence is corresponding to said cDNA sequence) that causes of process.Can design the dsRNA molecule so that make each residue and the residue in the target molecule in the antisense strand complementary.Perhaps, can replace with stability that increases said molecule and/or the processing activity that strengthens said molecule at intramolecularly.Can in chain, replace, maybe can the terminal residue of chain be replaced.In certain embodiments, the specific residue place in DsiRNA reagent replaces and/or modifies.This type replaces and/or modifies and can comprise, for example, when from 3 ' end position numbering of the sense strand of DsiRNA reagent, the deoxidation that one or more residues place of 1,2 and 3 carries out in the position is modified; The deoxidation that one or more residues of 1,2,3 or 4 carry out in the position when from 5 ' end position numbering of the antisense strand of DsiRNA reagent is modified; And introduce at 3 of the antisense strand of DsiRNA reagent ' end residue place '-the O-alkyl is (for example; 2 '-the O-methyl) modify; Wherein this type modification likewise or alternatively is present in the extrusion position place of 3 of antisense strand ' part and/or is present in DsiRNA reagent everywhere; For example, the alternately residue or the paired residue place that are present in the DsiRNA antisense strand that comprises in the district of the active siRNA reagent of being processed to form of DsiRNA reagent.The modification of front provides as an example, and does not plan to limit by any way.The further considering of structure of relevant preferred DsiRNA reagent be can find hereinafter, modification and substituted the further describing that to implement DsiRNA reagent of the present invention comprised.
So-called " complementation " is meant that nucleic acid can form hydrogen bond through traditional Watson-Crick or other non-traditional format with another nucleotide sequence.About nucleic acid molecules of the present invention, nucleic acid molecules is enough to make nucleic acid to fulfil correlation function with the free energy that combines of its complementary series, and for example, RNAi is active.The combination free energy of measuring nucleic acid molecules be well known in the art (for example, referring to Turner etc., 1987, CSH Symp.Quant.Biol.LII, 123-133 page or leaf; Frier etc., 1986, Proc.Nat.Acad.Sci.USA 83:9373-9377; Turner etc., 1987, J.Am.Chem.Soc.109:3783-3785).Complementary percentage ratio is indicated in a certain nucleic acid molecules can (for example form hydrogen bond with second nucleotide sequence; The percentage rate of the continuous residue Watson-Crick base pairing) (for example, having 5,6,7,8,9 or 10 nucleotide and the second nucleotide sequence base pairing to represent 50%, 60%, 70%, 80%, 90% and 100% complementation respectively among 10 nucleotide altogether in first oligonucleotide) with 10 nucleotide." complementary fully " is meant that all the continuous residues in a certain nucleotide sequence will form hydrogen bond with the continuous residue of similar number in second nucleotide sequence.In one embodiment; DsiRNA molecule of the present invention comprises and complementary about 19 to about 30 of one or more target nucleic acid molecules or its parts (for example, about 19,20,21,22,23,24,25,26,27,28,29 or 30 or more a plurality of) nucleotide.
Phrase " double-stranded tagma " is meant the district in two complementations or the complementary basically oligonucleotide, and said oligonucleotide is through the Watson-Crick base pairing or allow any alternate manner of the formation duplex between complementary or the complementary basically oligonucleotide chain to form base pair each other.For example, have 21 nucleotide units oligonucleotide chain can with another oligonucleotide base pairing with 21 nucleotide units, but 19 base complementrities or complementary basically only on each chain are so that " double-stranded tagma " is made up of 19 base pairs.The residue base pair can be for example as 5 ' with 3 ' outstanding the existence.In addition, in double-stranded tagma, do not need 100% complementation; Content is permitted complementary basically in double-stranded tagma.
Basically complementary be meant between the chain complementation so that its can under biotic factor, anneal.Confirm by rule of thumb whether two chains can annealed technology be well known in the art under biotic factor.Perhaps, can under biotic factor, two chains be synthesized and add together, to confirm the whether each other annealing of two chains.
Single-chain nucleic acid carries out base pairing in some bases and is called as " hybridization ".Hybridization usually on the physiology or biologically relevant condition (for example, in the cell: pH 7.2, the 140mM potassium ion; Extracellular: pH7.4, the 145mM sodium ion) confirm down.Hybridization conditions contains monovalent cation and biologically acceptable buffer usually; And said hybridization conditions can contain or can (for example not contain divalent cation, complex anion; Glucose acid group from potassium gluconate), uncharged material (such as; Sucrose) and reduce sample water active inert polymer (for example, PEG) in.This type condition comprises the condition that can form base pair.
The required temperature of single-chain nucleic acid that forms duplex (is a melting temperature through dissociating; Tm) tolerance hybridization.Hybridization conditions also is the condition that can form base pair.Can use various stringent conditions with measure hybridization (for example, referring to Wahl, G.M. and S.L.Berger (1987) Methods Enzymol.152:399; Kimmel, A.R. (1987) Methods Enzymol.152:507).Strict temperature conditions will comprise at least about 30 ℃ usually, more preferably at least about 37 ℃, and most preferably at least about 42 ℃ temperature.The hybridization temperature that length is contemplated to less than the hybrid of 50 base pairs should hang down 5 to 10 ℃ than the melting temperature (Tm) of this hybrid, wherein confirms Tm according to following equality.Concerning length less than the hybrid of 18 base pairs, Tm (℃)=2 (quantity of A+T base)+4 (quantity of G+C base).Concerning length is the hybrid of 18 to 49 base pairs; Tm (℃)=81.5+16.6 (log10 [Na+])+0.41 (%G+C)-(600/N); Wherein N is the base number in the crossbred, and [Na+] is the concentration (is 0.165M for 1 * SSC [Na+]) of sodium ion in the hybridization buffer.For example, at the buffer of hybridization assays shown in the table 1.
Table 1.
Figure BDA0000133674250000321
The useful variation of hybridization conditions will be conspicuous to those skilled in the art.Hybridization technique is well-known to those skilled in the art, and is described in (for example) Benton and Davis (Science 196:180,1977); Grunstein and Hogness (Proc.Natl.Acad.Sci., USA 72:3961,1975); Ausubel etc. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Antisense to Molecular Cloning Techniques, 1987, Academic Press, New York); And Sambrook etc., Molecular Cloning:A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.
As used herein, " oligonucleotide chain " is single stranded nucleic acid molecule.Oligonucleotide can comprise ribonucleotide, deoxyribonucleotide, nucleotide (for example, having the 2 ' nucleotide of modifying, synthetic base analogue etc.) or its combination through modifying.This type can be superior to native form through the oligonucleotide of modifying, because it has for example enhanced cell picked-up and in the characteristic that has the stability that increases under the situation of nuclease.
Some dsRNA of the present invention can be chimeric double stranded RNA (dsRNA)." chimeric dsRNA " or " chimera " are for containing two or more chemically different districts dsRNA of (each district is made up of at least one nucleotide) under situation of the present invention.These dsRNA contain at least one usually and mainly comprise the ribonucleotide district of (randomly, comprising the ribonucleotide through modifying), and said ribonucleotide forms Dicer substrate siRNA (" DsiRNA ") molecule.This DsiRNA district can be covalently bound to base pairing deoxyribonucleotide (" dsDNA district ") second district that comprises on the arbitrary side that is arranged in the double-stranded tagma of ribonucleotide; This (for example can give one or more beneficial characteristics; For example improve effect; Improve active effectiveness of DsiRNA and/or persistent period, as the recognition structure territory that makes chimeric dsNA targeting specific position (for example, when to cells in culture or when the curee uses) or method, as improve region of elongation that functional group, payload, detection/detectable moiety connect, as the better modification and/or the improvement region of elongation at interval of this type modification etc. are provided).This second district (for example, comprising the base pairing deoxyribonucleotide) also can comprise to be modified or synthetic nucleotide and/or modification or synthetic deoxyribonucleotide.
As used herein, that term " ribonucleotide " is contained is natural and synthetic, without modifying and ribonucleotide through modifying.Modification comprises the change to the bonding between sugar moieties, base portion and/or the ribonucleotide in the oligonucleotide.As used herein, term " ribonucleotide " does not especially comprise deoxyribonucleotide, and it is for having the nucleotide of single proton group in 2 ' ribose ring position.
As used herein, that term " deoxyribonucleotide " is contained is natural and synthetic, without modifying and deoxyribonucleotide through modifying.Modification comprises to sugar moieties in the oligonucleotide, to base portion and/or to the change of bonding between the deoxyribonucleotide.As used herein; Term " deoxyribonucleotide " also comprises the ribonucleotide through modifying; The ribonucleotide that this process is modified (does not for example allow dsRNA reagent; 2 '-ribonucleotide residue that O-methyl ribonucleotides, D2EHDTPA are modified etc.) the Dicer cracking, saidly do not allow the Dicer cracking to come across the bonding place of this residue through the ribonucleotide of modifying.
As used herein, term " PS-NA " is meant the nucleotide residue that D2EHDTPA is modified.Therefore, the ribonucleotide (" PS-RNA ") of D2EHDTPA modification and the deoxyribonucleotide (" PS-DNA ") that D2EHDTPA is modified contained in term " PS-NA ".
As used herein; " Dicer " is meant the endoribonuclease in the rnase iii family; This endoribonuclease is with dsRNA or contain dsRNA molecule (for example, double-stranded RNA (dsRNA) or Microrna precursor (miRNA)) and be cracked into length and on 3 ' end, have the double stranded nucleic acid fragment that two bases are given prominence to usually for about 19-25 nucleotide.With regard to dsRNA of the present invention, the duplex that is formed by the dsRNA district of dsRNA of the present invention is through Dicer identification, and at least one chain of duplex, is the Dicer substrate.The first step in the Dicer catalysis RNA interference channel, thus the degraded of target RNA caused.The protein sequence of people Dicer is provided in the ncbi database under the number of the landing NP_085124, and this ncbi database is incorporated at this by reference.
Measure Dicer " cracking " (for example, referring to Collingwood etc., Oligonucleotides18:187-200 (2008)) by being described below.In the Dicer cracking is measured; At the recombined human Dicer (Stratagene that has or do not exist 1 unit; La Jolla, under situation CA) under 37 ℃ with 20mM Tris (pH 8.0), 200mM NaCl, the 2.5mM MgCl of RNA duplex (100pmol) at 20 μ L 2In hatched 18 to 24 hours.(Edge Biosystems, Gaithersburg is MD) with the sample desalination to use Performa SR 96 orifice plates.Use Oligo HTCS system (Novatia, Princeton, NJ; Hail etc.; 2004) before Dicer handles with after handling, duplex RNA is carried out electron spray ionisation liquid chromatography mass analysis (ESI-LCMS); Said Oligo HTCS system is by ThermoFinnigan TSQ7000, Xcalibur data system, ProMass data processing software and Paradigm MS4HPLC (Michrom BioResources; Auburn CA) forms.The Dicer cracking takes place in this measures, wherein at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or even 100% Dicer substrate dsRNA (that is 25-30bp; DsRNA; Preferably, 26-30bp dsRNA) be cracked into shorter dsRNA (for example, 19-23bp dsRNA; Preferably, 21-23bp dsRNA).
As used herein, " Dicer cracking site " is meant the site (for example, the dsRNA district of dsRNA of the present invention) of Dicer cracking dsRNA.Dicer contains the sense strand of common cracking dsRNA and two rnase iii domains of antisense strand.The length of the said short double stranded nucleic acid fragment that it is produced of average distance decision between rnase iii domain and the PAZ domain; And this distance can change (Macrae I etc., (2006). " Structural basis for double-stranded RNA processing by Dicer " .Science 311 (5758): 195-8).Estimate Dicer cracking some double-strandednucleic acid of the present invention, the antisense strand that these double-strandednucleic acids have is for away from the site between the 21st of 3 of antisense strand ' end and the 22nd nucleotide and have 3 of 2 nucleotide ' give prominence to away from the corresponding site between the 21st of sense strand 5 ' end and the 22nd nucleotide.The dsRNA molecule estimate and/or general Dicer cracking site different with Dicer cracking site known in the art, or can discern through art-recognized method (comprise Macrae etc. described in those methods) similarly.The Dicer cracking of dsRNA (for example, DsiRNA) can cause producing the siRNA length through Dicer processing that length is 19 to 23 nucleotide.In fact, in greater detail in one aspect of the present invention, the double-stranded DNA district is included in the dsRNA, generally excises not preferred 19mer siRNA to instruct common Dicer hereinafter.
As used herein, " giving prominence to " is meant at 5 of dsRNA ' end or 3 ' end place to have unpaired nucleotide under the situation of one, two, three, four or five free-ended duplex.In certain embodiments, outstandingly be 3 on antisense strand or the sense strand ' or 5 ' outstanding.
As used herein; Term " DmiRNA " is meant that (especially in the district of antisense strand) in a kind of antisense at DmiRNA reagent (guiding) chain has the Dicer substrate siRNA (" DsiRNA ") of at least one mispairing nucleotide, and said Dicer substrate siRNA works as rnai agent and is considered to the sequence hybridization with target RNA.With regard to regard to justice (passerby) chain is arranged, just can have this mispairing nucleotide with regard to both with regard to the target RNA sequence (antisense strand of DmiRNA is considered to and said target RNA sequence hybridization) or with regard to above.
As used herein; Term " RNA processing " by the component of siRNA, miRNA or ribonuclease H approach (for example is meant; Drosha, Dicer, Argonaute2 or other RISC endoribonuclease and ribonuclease H) the processing activity carried out, this has made more detailed description (" RNA processing " part vide infra) hereinafter.Said term clearly is different from 5 of the RNA that carries out through the processing of the processing of non-RISC mediation or the mediation of non-ribonuclease H ' and adds the post-treatment of transcribing that medicated cap and RNA degrade.RNA this " degraded " can be adopted some forms; For example deadenylation (remove 3 ' poly A tail) and/or any through in some Cobra venom endonucleases or the exonuclease (for example, rnase iii, ribonuclease P, ribonuclease T1, ribonuclease A (1,2,3,4/5), oligonucleotidase etc.) with partly or entirely carrying out nuclease digestion in the RNA main body.
" homologous sequence " be meant by one or more polynucleotide sequences (such as, gene, genetic transcription thing and/or non-coded polynucleotide) total nucleotide sequence.For example; Homologous sequence can serve as reasons two or more for relevant but different protein (such as; Different members in the gene family, different proteins epi-position, different proteins isotype or diverse gene (such as, the cytokine receptor corresponding) with its) the common nucleotide sequence of gene of coding.Homologous sequence can serve as reasons two or more non-coded polynucleotides (such as, noncoding DNA or RNA, regulating and controlling sequence, intron and transcribe control or regulatory site) common nucleotide sequence.Homologous sequence can also comprise by a conserved sequence district that above polynucleotide sequence is common.Homology (for example needs not to be complete homology; 100%); Because homeologous sequence (for example, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80% etc.) is also contained in the present invention.In fact; The probability of using this DsiRNA reagent is contained in the design and use of DsiRNA reagent of the present invention; Said DsiRNA reagent not only to and the complete complementary target RNA of present said DsiRNA reagent, but also to having and the said DsiRNA reagent target RNA of complementary sequence such as (for example) 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80% only.Similarly; The expection technical staff can easily change the present described DsiRNA reagent of the present invention; To strengthen said DsiRNA reagent and the target RNA of institute (for example, the complementary degree between the specific alleles variant (for example, having the allele that strengthens the treatment benefit).In fact, the DsiRNA reagent sequence that has insertion, disappearance and a simple point mutation with respect to the target sequence can also suppress (possibly be considered to suppress rather than destroy to work through the Microrna appearance translation of target transcript effectively; Therefore, this DsiRNA reagent can be described as " DmiRNA ").Perhaps, the DsiRNA reagent sequence that has nucleotide analog replacement or insertion can suppress effectively.
Can measure sequence homogeneity through sequence comparison known in the art and alignment algorithm.In order to measure the homogeneity percentage ratio of two nucleotide sequences (or two aminoacid sequences), for the comparison purpose of optimum (for example, can first sequence be introduced or second sequence is compared for optimum in the room) aligned sequences.Subsequently, the nucleotide (or amino acid residue) of more corresponding nucleotide (or aminoacid) position.When the residue of the residue that occupies a certain position of first sequence and the second sequence relevant position was identical, then molecule was identical in this position.Homogeneity percentage ratio between two sequences is the function (that is, % homology=same position quantity/total number of positions multiply by 100) of the common same position number of sequence, randomly carries out point penalty to the quantity of introducing the room and/or the length of introducing the room.
Can use mathematical algorithm to accomplish the mensuration of sequence homogeneity percentage ratio relatively and between two sequences.In one embodiment, in certain part of the aligned sequences with enough homogeneity, compare, and in the part that has than the homogeneity of low degree, do not compare (that is local comparison).Preferred, the limiting examples that are used for the local alignment algorithm of sequence comparison are algorithm (1990) the Proc.Natl.Acad.Sci.USA 87:2264-68 of Karlin and Altschul, and said algorithm is revised in Karlin and Altschu (1993) Proc.Natl.Acad.Sci.USA 90:5873-77.This algorithm is incorporated in the blast program (2.0 version) of (1990) J.Mol.Biol.215:403-10 such as Altschul.
In another embodiment, make comparison optimization, and measure the interior homogeneity percentage ratio (that is room comparison) of length of aligned sequences through introducing suitable room.In order to obtain to be used for the room comparison of comparison purpose, can use like Altschul etc., (1997) Nucleic Acids Res.25 (17): the Gapped BLAST described in the 3389-3402.In another embodiment, make comparison optimization, and measure the interior homogeneity percentage ratio (that is whole comparison) of whole length of aligned sequences through introducing suitable room.The preferred limiting examples of mathematical algorithm that is used for the integral body comparison of sequence is the algorithm of Myers and Miller, CABIOS (1989).This algorithm is incorporated in the ALIGN program (2.0 version), and this ALIGN program is the part of GCG sequence alignment software kit.When using the ALIGN program to come the comparing amino acid sequence, can use PAM120 weight residue table, room length point penalty 12 and gap penalty 4.
Sequence homogeneity between the part in preferred DsiRNA antisense strand and the target RNA sequence is greater than 80%; For example, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even 100% sequence homogeneity.Perhaps, can DsiRNA be defined as on function can (for example, 400mM NaCl, 40mM PIPES pH 6.4,1mM EDTA be in 50 ℃ or 70 ℃ hybridization 12 to 16 hours down with the hybridization of the part of target RNA; Wash subsequently) nucleotide sequence (or oligonucleotide sequence).Preferred hybridization conditions in addition comprises: in 1 * SSC, hybridize down at 50 ℃ down or in 1 * SSC, 50% Methanamide at 70 ℃; In 0.3 * SSC, wash down subsequently at 70 ℃; Perhaps in 4 * SSC, hybridize down at 50 ℃ down or in 4 * SSC, 50% Methanamide, in 1 * SSC, wash down subsequently at 67 ℃ at 70 ℃.Expection length should be hanged down 5 to 10 ℃ than the melting temperature (Tm) of hybrid less than the hybridization temperature of the hybrid of 50 base pairs, wherein confirms Tm according to following equality.Concerning length less than the hybrid of 18 base pairs, Tm (℃)=2 (quantity of A+T base)+4 (quantity of G+C base).Concerning length is the hybrid of 18 to 49 base pairs; Tm (℃)=81.5+16.6 (log10 [Na+])+0.41 (%G+C)-(600/N); Wherein N is the base number in the hybrid, and [Na+] is the concentration (is 0.165M for 1 * SSC [Na+]) of sodium ion in the hybridization buffer.The other instance that is used for the stringent condition of multi-nucleotide hybrid is provided in Sambrook, J., E.F.Fritsch and T.Maniatis; 1989, Molecular Cloning:A Laboratory Manual, Cold Spring Harbor Laboratory press; Cold Spring Harbor, N.Y., the 9th Zhanghe Chapter 11; With Current Protocols in Molecular Biology, 1995, volumes such as F.M.Ausubel; John Wiley&Sons, Inc. is among chapters and sections 2.10 and the 6.3-6.4.The length of identical nucleotide sequence can be at least about 10,12,15,17,20,22,25,27 or 30 bases.
" conserved sequence district " is meant that the nucleotide sequence in one or more districts in the polynucleotide does not significantly change between the generation or from living things system, curee or an organism to another living things system, curee or organism.Polynucleotide can comprise coding and noncoding DNA and RNA.
" the justice district is arranged " and be meant the nucleotide sequence that has complementary DsiRNA molecule with the antisense district of DsiRNA molecule.In addition, the adopted district that has of DsiRNA molecule can comprise the nucleotide sequence that has homology with target nucleic acid sequence.
" antisense district " is meant the nucleotide sequence that has complementary DsiRNA molecule with target nucleic acid sequence.In addition, the antisense district of DsiRNA molecule comprises and the adopted nucleotide sequence with complementarity of distinguishing of having of DsiRNA molecule.
As used herein, " antisense strand " is meant the single stranded nucleic acid molecule that has with the complementary sequence of target RNA sequence.When antisense strand contained the nucleotide of the process modification with base analogue, said antisense strand may not be complementary in whole length, but must hybridize with target RNA at least.
As used herein, " sense strand " is meant the single stranded nucleic acid molecule that has with the complementary sequence of antisense strand sequence.When antisense strand contains when having the nucleotide that advancing of base analogue modify, sense strand may not be complementary in the whole length of said antisense strand, but must form duplex with antisense strand at least.
As used herein, " guiding chain " is meant dsRNA or contains the single stranded nucleic acid molecule of dsRNA molecule, and this single stranded nucleic acid molecule has with target RNA sequence fully complementary to produce the RNA interference.After making dsRNA through Dicer or containing the cracking of dsRNA molecule, guiding chain fragment keeps being connected with RISC, combine to pass through the RISC cracking as the target RNA and the promotion target RNA of the component of RISC complex.As used herein, the guiding chain may not refer to successive single-chain nucleic acid, and said guiding chain can comprise discontinuity, preferably by the cracked site of Dicer.The guiding chain is an antisense strand.
As used herein, " passerby's chain " is meant dsRNA or contains the oligonucleotide chain of dsRNA molecule, and this oligonucleotide chain has and the complementary sequence of guiding chain-ordering.As used herein, passerby's chain may not refer to successive single-chain nucleic acid, and can comprise discontinuity, preferably by the cracked site of Dicer.Passerby's chain is a sense strand.
" target nucleic acid " is meant its expression, level or the active any nucleotide sequence that remains to be regulated.Target nucleic acid can be DNA or RNA.Also can come the targeted expression level, or the molecule in downstream that also can be through targeting (for example) target target signal transduction pathway is regulated through overregulating or the effect of the wrong target of regulating through the upper reaches effector of targeting target.
Known, the RNAi method is applicable to the multiple gene in the multiple organism, and disclosed compositions and method can be used for each in these situation.The instance of gene that can be through disclosed compositions and method targeting comprises endogenous gene, and said endogenous gene is the primary gene of cell or is not the primary gene of cell usually.Non-exclusively; These genes comprise the gene, cell surface receptor gene, the GFP that relates to transfer, protease gene, apoptosis gene, cell cycle controlling gene, the gene of expressing EGF and EGF receptor, multidrug-resisting gene (such as, MDR1 gene) of gene, the adhesion molecule of the molecule of oncogene, cytokine gene, idiotype (Id) GFP, prion protein gene, induced expression angiogenesis.
More particularly, the aminoacid sequence of said target mrna designated cell protein of the present invention (for example, nucleus, Cytoplasm, stride film or embrane-associated protein).In another embodiment, the aminoacid sequence of the outer albumen (for example, extracellular matrix protein or secretory protein) of said target mrna designated cell of the present invention.As used herein, phrase " is specified proteinic aminoacid sequence " and is meant that the rule according to genetic code is translated as aminoacid sequence with the mRNA sequence.List following protein classification and be used for illustrative purposes: grow albumen (for example, adhesion molecule, cyclin kinase inhibitor, Wnt family member, Pax family member, wing spiral family member, Hox family member, cytokine/lymphokine and their receptor, growth/differentiation factor and their receptor, neurotransmitter and their receptor); Oncogene encoded protein matter (for example, ABLI, BCLI, BCL2, BCL6, CBFA2, CBL, CSFIR, ERBA, ERBB, EBRB2, ETSI, ETSI, ETV6, FGR, FOS, FYN, HCR, HRAS, JUN, KRAS, LCK, LYN, MDM2, MLL, MYB, MYC, MYCLI, MYCN, NRAS, PIMI, PML, RET, SRC, TALI, TCL3 and YES); Tumor suppressor protein (for example; BRCA1, BRCA2, MADH4, MCC, NFI, NF2, RBI, TP53 and WTI) and enzyme (for example, acc synthase and oxidase, ACP desaturase and hydroxylase, ADP glucose phosphorylation enzyme, ATP enzyme, ethanol dehydrogenase, amylase, amyloglucosidase, catalase, cellulase, chalcone synthase, chitinase, cyclooxygenase, decarboxylase, dextromase, DNA and RNA polymerase, tilactase, glucanase, glucoseoxidase, granule combine starch synthase, GTP enzyme, unwindase, hemicellulase, intergrase, inulinase, invertase, isomerase, kinases, Lactose enzyme, lipase, lipoxidase, lysozyme, nopaline synthase, octopine synthase, pectinesterase, peroxidase, phosphatase, phospholipase, phosphorylase, phytase, plant growth regulator synthase, polygalacturonase, protease and peptidase, pullulanase, recombinase, reverse transcriptase, rubisco, topoisomerase and xylanase), ApoB100 and HPRT1.
In one aspect, the aminoacid sequence of said target mrna molecule appointment of the present invention and pathological state proteins associated matter.For example; Protein (for example can be the protein relevant with pathogen; Relate to host's the duplicating of immunosuppressant, pathogen, the propagation of pathogen or the virus protein of keeping of infection) or help pathogen to get into the host, duplicate or integrate, infect foundation or the diffusion in the host, the perhaps host protein of the assembling of pathogen of future generation through what pathogen or host carried out drug metabolism, pathogen gene group.Pathogen comprises RNA viruses; Such as banzi virus, picornavirus, rhabdovirus, filamentous form virus, retrovirus (comprising lentivirus) or DNA viruses; Such as, adenovirus, poxvirus, herpesvirus, cytomegalovirus, hepadnavirus or other virus.Other pathogen comprises antibacterial, fungus, anthelmintic, schistosomicide and trypanosoma.The pathogen of other kind can comprise the mammal transposable element.Perhaps, protein can be tumor correlated albumen or and autoimmune disease associated protein.
Target gene can be derived from or be contained in any organism.Organism can be plant, animal, protozoacide, antibacterial, virus or fungus.For example referring to United States Patent (USP) the 6th, 506, No. 559, it incorporates this paper by reference into.
In one embodiment of the invention; The length of each sequence of DsiRNA molecule of the present invention is about 25 to about 35 nucleotide independently, is 25,26,27,28,29,30,31,32,33,34 or 35 nucleotide in specific embodiments on length.In another embodiment, DsiRNA duplex of the present invention comprises about 25 independently to about 30 base pairs (for example, about 25,26,27,28,29 or 30).In another embodiment; One or more chain of DsiRNA molecule of the present invention comprises and target nucleic acid molecule complementary about 19 to about 35 nucleotide (for example, about 19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34 or 35) independently.At Fig. 1 with exemplary DsiRNA molecule of the present invention hereinafter is shown.
As used herein, " cell " uses with its common biological meaning, and is not meant whole multicellular organisms, for example, is not meant the people specifically.Cell can be present in the organism, for example birds, plant and mammal, such as, people, cattle, sheep, ape, monkey, pig, Canis familiaris L. and cat.Cell can be (for example, the mammalian cell or the plant cell) of protokaryon (for example, bacterial cell) or eucaryon.It is the source or the system genitale source, all-round or polyenergic, splitted or not splitted that cell can be somatic cell.Cell can also be derived from the cell that maybe can comprise gamete or embryo, stem cell or break up fully.In some aspects, the mammalian cell of the separation dsNA molecule one or more of the present disclosure that term " cell " specifically refers to contain, such as, people's cell.In particular aspects, cell is processed dsRNA or is contained the dsRNA molecule and causes the RNA of target nucleic acid to disturb, and said cell contains required protein of RNAi and protein complex, for example Dicer and RISC.
DsiRNA molecule of the present invention is directly added target cell or tissue, maybe can DsiRNA molecule of the present invention and cation lipid is compound, be wrapped in the liposome, perhaps otherwise be delivered to target cell or tissue.Can in external or body, through direct cutaneous application, percutaneous application or injection nucleic acid or nucleic acid complexes be locally applied to linked groups, its amplifying nucleic acid or nucleic acid complexes are incorporated biopolymer into or are not incorporated biopolymer into.Of the present invention aspect some, according to the modification pattern of following described DsiRNA reagent, the dsRNA of the exemplary configurations of the dsRNA peptide that Fig. 1 is appeared modifies.The construct of the form of chemical modification described in Fig. 1 and following exemplary configurations can be used in DsiRNA reagent as herein described described any and all purposes.
In yet another aspect, the present invention provides the mammalian cell that contains one or more DsiRNA molecules of the present invention.Can be with one or more DsiRNA molecules targeting same loci or different loci independently.
So-called " RNA " is meant the molecule that comprises at least one ribonucleotide residue.Said " ribonucleotide " is meant in 2 ' position of β-D-ribofuranose part to have the nucleotide of hydroxyl.Said term comprises double-stranded RNA, single stranded RNA, isolation of RNA; Such as; Partial purification RNA, the RNA that produces of purifying RNA, synthetic RNA, reorganization basically, and the change RNA that is different from natural RNA that forms of interpolation, disappearance, replacement and/or change through one or more nucleotide.This type of change can comprise for example terminal or inner to DsiRNA, for example adds the non-nucleotide material at one or more nucleotide place of RNA.Nucleotide in the RNA molecule of the present invention can also comprise non-standard nucleotide, such as the nucleotide or the Deoxydization nucleotide of non-natural nucleotide or chemosynthesis.The RNA of these changes can be considered to the analog of analog or natural RNA.
Said " curee " is meant as the donor of outer explant cell or the organism or the cell itself of receptor." curee " also refers to use to it organism of DsiRNA reagent of the present invention.The curee can be mammal or mammalian cell, comprises people or people's cell.
Phrase " pharmaceutically acceptable carrier " is meant the carrier that is used for the administering therapeutic agent.Exemplary carrier comprises saline, BS, glucose, water, glycerol, ethanol and combination thereof.Concerning Orally administered medicine, pharmaceutically acceptable carrier includes but not limited to pharmaceutically acceptable excipient, such as inert diluent, disintegrating agent, binding agent, lubricant, sweeting agent, flavoring agent, coloring agent and antiseptic.Suitable inert diluent comprises sodium carbonate and calcium carbonate, sodium phosphate and calcium phosphate and lactose, and corn starch and alginic acid are suitable disintegrants.Binding agent can comprise starch and gelatin, and lubricant (if existence) will be magnesium stearate, stearic acid or Talcum usually.If desired, tablet can scribble such as materials such as glyceryl monostearate or distearins, to postpone the absorption in intestines and stomach.The pharmaceutically acceptable carrier of disclosed dsRNA compositions can be micellar structure, such as liposome, capsid, virocapsomer (capsoid), polymer/nanometer capsule or polymerization microcapsule.
Polymer/nanometer capsule or microcapsule help dsRNA transportation encapsulation or bonded and are released in the cell.They comprise polymeric material and monomer material, especially comprise paracyanogen base butylacrylate.Delivered the general introduction (referring to Kreuter, 1991) of material and manufacturing approach.Producing the polymeric material itself that is formed by monomer and/or oligomerization precursor in the step in polymerization/nano-particle is that prior art is known, and the technical staff of manufacturing field of nanoparticles can be according to the common technology suitably molecular weight and the molecular weight distribution of selective polymerization material.
The whole bag of tricks of the present invention comprises and relates to the step that numerical value, level, characteristics, characteristic, characteristic etc. and " suitable contrast " (replacedly being called " suitable contrast " in this article) are compared." suitable contrast " or " suitable contrast " is any contrast or the standard that those of ordinary skill in the art knew for can be used for the comparison purpose.In one embodiment, " suitable contrast " or " suitable contrast " is as described herein in the numerical value that carries out being measured before the RNAi method, level, characteristics, characteristic, characteristic etc.For example, in the reticent agent with RNA of the present invention (confirmable transcription rate, mRNA level, translation speed, protein level, biological activity, cell characteristic or characteristic, genotype, phenotype etc. before for example, DsiRNA) introducing in cell or the organism.In another embodiment, " suitable contrast " or " suitable contrast " is the numerical value in the cell of the for example normal characteristic of performance or organism (for example, contrast or normal cell or organism), measured, level, characteristics, characteristic, characteristic etc.In another embodiment, " suitable contrast " or " suitable contrast " is predetermined numerical value, level, characteristics, characteristic, characteristic etc.
Term " external " has its art-recognized implication, for example relates to purified reagent or extract, for example, and cell extract.Term " in the body " also has its field art-recognized meanings, for example relates to the living cells in the organism, for example, and immortality cell, primary cell, cell line and/or cell.
Patient's application of as used herein " treatment " definition subtend or administering therapeutic agent are (for example; DsiRNA reagent or carrier or its transgenic of encoding) or use or the administering therapeutic agent to chorista or cell line from the patient who suffers from disease, with the purpose of the symptom that reaches treatment, cure, alleviate, alleviate, change, correct, improve, improve or influence disease or disease or disease or disease.Term " treatment " also is used for prophylactically using the situation of reagent in this article.Term " effective dose (effective dose) " or " effective dose (effective dosage) " be defined as be enough to reach or reach at least in part the amount that will act on.Term " treatment effective dose " is defined as the amount that is enough to treat or stop at least in part ill disease of patient and its complication.Term " patient " comprises the people and other mammalian subject who accepts preventative or therapeutic treatment.
The design of dsRNA-peptide/synthetic
Find by rule of thumb to compare with 19 to 23mer siRNA reagent, 25 to about 35 nucleotide (DsiRNA) and especially be 25 to the longer dsRNA kinds of about 30 nucleotide with regard to render a service and persistent period of effect with regard to provided beat all effective result.Under the situation of the potential theory of not hoping to receive the dsRNA processing mechanism, think that longer dsRNA kind is used as the substrate of Dicer enzyme in the Cytoplasm of cell.Dicer thinks also that except dsRNA cracking of the present invention is become than the short segments Dicer helps the strand pyrolysis product that derives from cracking dsRNA is incorporated in the RISC complex of being responsible for destruction target RNA.Existing research (Rossi etc., No. the 2007/0265220th, U.S. Patent application) has shown that dsRNA kind (DsiRNA reagent specifically) is consistent with the raising effectiveness and the acting duration of dsRNA kind through the cleavable property of Dicer.
The dsRNA that comprises double-stranded RNA is contained in the present invention; Said double-stranded RNA comprises first chain and second chain; Wherein the length of first chain and second chain is at least 16 and 50 nucleotide (for example, length is 16 to 50 nucleotide, 19 to 35 nucleotide, 19 to 24 nucleotide, 25 to 30 nucleotide, 25,35,19 to 23 nucleotide and 21 to 23 nucleotide) at the most.
A.dsRNA
The design of dsRNA (comprising DsiRNA) can randomly relate to uses the prediction scoring algorithm, and this prediction scoring algorithm carries out expectation activity/effect that several possible DsiRNA reagent of sequence area are crossed in the computer simulation evaluation.For example; Can be at (BMC Bioinformatics 2006 such as Gong; Find the design information of relevant this type of scoring algorithm 7:516), but with respect to the previously used siRNA scoring algorithm in this area, the improved in theory algorithm of " v3 " algorithmic notation of renewal.(" v3 " scoring algorithm is not for relying on the machine learning algorithm of anyone ordering bias.In addition, the data set that derives of " v3 " algorithm be before about three times of the data set that derives of " v2 " algorithm (such as that algorithm described in the Gong etc.).)
First and second oligonucleotide of DsiRNA reagent of the present invention do not need complete complementation.In fact, in one embodiment, 3 of sense strand ' end contains one or more mispairing.In one aspect, incorporate about two mispairing at 3 ' end place of sense strand.In another embodiment; DsiRNA of the present invention is the double stranded rna molecule that contains two RNA oligonucleotide; The length of each said RNA oligonucleotide is 27 nucleotide, and when annealing each other, has flush end and two nucleotide mispairing on 3 of sense strand ' end (5 of antisense strand ' end).Advised using mispairing or reduction thermodynamic stability (especially sense strand 3 '/antisense strand 5 ' position); Probably to promote or to help antisense strand to get into RISC (Schwarz etc. through the step of untwisting (said step occurs along with siRNA gets into RISC) that influences some limiting speeds; 2003, Cell 115:199-208; Khvorova etc., 2003, Cell 115:209-216)).Therefore, the terminal bases compositions has been included in the algorithm for design, to select active 21mer siRNA duplex (Ui-Tei etc., 2004, Nucleic Acids Res 32:936-948; Reynolds etc., 2004, Nat Biotechnol 22:326-330).Because the Dicer cracking takes place for the dsRNA of this embodiment, the small end-end sequence that contains mispairing will keep leaving final 21mer siRNA with antisense strand unpaired (become 3 ' in outstanding a part) or complete cracking.Therefore, these " mispairing " can not continue to be retained in the final RNA component of RISC as mispairing.Discovery improves the effectiveness of synthesizing duplex among the RNAi through being promoted to process by Dicer by inference at segmental base mispairing in 3 ' end place of the sense strand of Dicer substrate or destabilization, and this discovery is to describe design and use 25 to 30mer dsRNA (to be called " DsiRNA " among this paper again; The accident of past work Rossi etc., U.S. Patent application No. 2005/0277610, No. 2005/0244858 and No. 2007/0265220) is found.
B. peptide
The present invention provides the compositions that comprises the dsRNA of the present invention that puts together with peptide.
Send peptide
In certain embodiments, target peptide is the defined peptides of sending of preceding text.
According to the useful peptide sequence of sending of the present invention
Compare with unconjugated dsRNA, according in the delivery rate of the acting duration of the useful dsRNA that sends peptide and increased the onset time of dsRNA, sent of the present invention or dsRNA of the present invention at least one.Reduce onset time like defined peptide of the present invention among this paper, make and compare with unconjugated dsRNA, reduce the lag time that target dsRNA arrives before the target RNA.Such as among this paper definition, the useful peptide of sending according to the present invention increases acting duration, makes and compare with unconjugated dsRNA that the dsRNA-peptide conjugate suppresses target RNA in the longer period.Such as among this paper definition, according to the useful delivery rate that peptide improves dsRNA of sending of the present invention, make the dsRNA-peptide conjugate arrive target RNA sooner than unconjugated dsRNA.
According to the present invention, measure and send the aminoacid sequence of peptide and it is optimized to be used to dsRNA to be sent.Be described in the document sending the useful peptide sequence of peptide according to the present invention.
In one embodiment; The peptide of sending according to the present invention comprises proline residue, for example, and sequence x1-P-x2-P-x3; Wherein x1 and x3 comprise 2 to 50 amino acid whose any aminoacid or fragments of peptides, and x2 is 0 or 1 aminoacid or contains 2 to 20 amino acid whose fragments of peptides.In another embodiment, x1=comprises the peptide of 5 amino acid residues; X2=comprises the peptide of 7 amino acid residues and the peptide that x3=comprises 4 amino acid residues.In another embodiment, x1=comprises the peptide of 8 amino acid residues; X2=comprises the peptide of 7 amino acid residues and the peptide that x3=comprises 4 amino acid residues.In another embodiment, x1=comprises the peptide of 8 amino acid residues; X2=comprises the peptide of 8 amino acid residues and peptide (Deber etc., Arch Biochem Biophys.1986 that x3=comprises 4 amino acid residues; 251 (1): 68-76; With Du etc., J Pept Res.1998; 51 (3): 235-43).
Being used for the peptide sequence of sending of the present invention includes but not limited to:
VRGIITSKTKSLDKGYNKALNDL(SEQ?ID?NO：1)
VRGIIPFKTKSLDEGYNKALNDL(SEQ?ID?NO：2)
KSVKAPGI(SEQ?ID?NO：3)
HKAIDGRSLYNKTLD(SEQ?ID?NO：4)
LRLTKNSRDDST(SEQ?ID?NO：5)
KNIVSVKGIRKSI(SEQ?ID?NO：6)
KSVIPRKGTKAPPRL(SEQ?ID?NO：7)
KPVMYKNTGKSEQ(SEQ?ID?NO：8)
EFVMNPANAQGHTPGTRL(SEQ?ID?NO：9)
EFVMNPANAQGHTAGTRL(SEQ?ID?NO：10)
EFVMNAANAQGHTPGTRL(SEQ?ID?NO：11)
EFVMNPANAQGRHTPGTRL(SEQ?ID?NO：12)
NPKEFVMNPANAQGHTPGTRL(SEQ?ID?NO：13)
NPKEFVMNPANAQGRHTPGTRL(SEQ?ID?NO：14)
KKIIPPTNIRENLYNRTASLTDLGGEL(SEQ?ID?NO：15)
CVRGIITSKTKSLDKGYNKALNDL(SEQ?ID?NO：16)
CVRGIIPFKTKSLDEGYNKALNDL(SEQ?ID?NO：17)
CKSVKAPGI(SEQ?ID?NO：18)
CHKAIDGRSLYNKTLD(SEQ?ID?NO：19)
CLRLTKNSRDDST(SEQ?ID?NO：20)
CKNIVSVKGIRKSI(SEQ?ID?NO：21)
CKSVIPRKGTKAPPRL(SEQ?ID?NO：22)
CKPVMYKNTGKSEQ(SEQ?ID?NO：23)
CEFVMNPANAQGHTPGTRL(SEQ?ID?NO：24)
CEFVMNPANAQGHTAGTRL(SEQ?ID?NO：25)
CEFVMNAANAQGHTPGTRL(SEQ?ID?NO：26)
CEFVMNPANAQGRHTPGTRL(SEQ?ID?NO：27)
CNPKEFVMNPANAQGHTPGTRL(SEQ?ID?NO：28)
CNPKEFVMNPANAQGRHTPGTRL(SEQ?ID?NO：29)
CKKIIPPTNIRENLYNRTASLTDLGGEL(SEQ?ID?NO：30)
GVRGIITSKTKSLDKGYNKALNDL(SEQ?ID?NO：31)
GVRGIIPFKTKSLDEGYNKALNDL(SEQ?ID?NO：32)
GKSVKAPGI(SEQ?ID?NO：33)
GHKAIDGRSLYNKTLD(SEQ?ID?NO：34)
GLRLTKNSRDDST(SEQ?ID?NO：35)
GKNIVSVKGIRKSI(SEQ?ID?NO：36)
GKSVIPRKGTKAPPRL(SEQ?ID?NO：37)
GKPVMYKNTGKSEQ(SEQ?ID?NO：38)
GEFVMNPANAQGHTPGTRL(SEQ?ID?NO：39)
GEFVMNPANAQGHTAGTRL(SEQ?ID?NO：40)
GEFVMNAANAQGHTPGTRL(SEQ?ID?NO：41)
GEFVMNPANAQGRHTPGTRL(SEQ?ID?NO：42)
GNPKEFVMNPANAQGHTPGTRL(SEQ?ID?NO：43)
GNPKEFVMNPANAQGRHTPGTRL(SEQ?ID?NO：44)
GKKIIPPTNIRENLYNRTASLTDLGGEL(SEQ?ID?NO：45)
VRGIITSKTKSLDKGYNKALNDLC(SEQ?ID?NO：46)
VRGIIPFKTKSLDEGYNKALNDLC(SEQ?ID?NO：47)
KSVKAPGIC(SEQ?ID?NO：48)
HKAIDGRSLYNKTLDC(SEQ?ID?NO：49)
LRLTKNSRDDSTC(SEQ?ID?NO：50)
KNIVSVKGIRKSIC(SEQ?ID?NO：51)
KSVIPRKGTKAPPRLC(SEQ?ID?NO：52)
KPVMYKNTGKSEQC(SEQ?ID?NO：53)
EFVMNPANAQGHTPGTRLC(SEQ?ID?NO：54)
EFVMNPANAQGHTAGTRLC(SEQ?ID?NO：55)
EFVMNAANAQGHTPGTRLC(SEQ?ID?NO：56)
EFVMNPANAQGRHTPGTRLC(SEQ?ID?NO：57)
NPKEFVMNPANAQGHTPGTRLC(SEQ?ID?NO：58)
NPKEFVMNPANAQGRHTPGTRLC(SEQ?ID?NO：59)
KKIIPPTNIRENLYNRTASLTDLGGELC(SEQ?ID?NO：60)
KSVKAPGIGGKSVKAPGI(SEQ?ID?NO：61)
KSVKAPGIGGKSVKAPGIGGKSVKAPGI(SEQ?ID?NO：62)
KSVKAPGIGG(KSVKAPGI) 2(SEQ?ID?NO：63)
CKSVKAPGIGGKSVKAPGI(SEQ?ID?NO：64)
CKSVKAPGIGGKSVKAPGIGGKSVKAPGI(SEQ?ID?NO：65)
GLFGAIAGFIENGWEGMIDGWYG(SEQ?ID?NO：66)
CGLFGAIAGFIENGWEGMIDGWYG(SEQ?ID?NO：67)
GLFGAIAGFIENGWEGMIDGWYGC(SEQ?ID?NO：68)
GRGDGG(SEQ?ID?NO：69)
CRGDGG(SEQ?ID?NO：70)
GRGDGC(SEQ?ID?NO：71)
THALWHT(SEQ?ID?NO：72)
GTHALWHT(SEQ?ID?NO：73)
THALWHTG(SEQ?ID?NO：74)
CTHALWHT(SEQ?ID?NO：75)
THALWHTC(SEQ?ID?NO：76)
QPFMQCLCLIYDASC(SEQ?ID?NO：77)
GQPFMQCLCLIYDASC(SEQ?ID?NO：78)
QPFMQCLCLIYDASCG(SEQ?ID?NO：79)
RNVPPIFNDVYWIAF(SEQ?ID?NO：80)
GRNVPPIFNDVYWIAF(SEQ?ID?NO：81)
RNVPPIFNDVYWIAFG(SEQ?ID?NO：82)
CRNVPPIFNDVYWIAF(SEQ?ID?NO：83)
RNVPPIFNDVYWIAFC(SEQ?ID?NO：84)
VFRVRPWYQSTSQS(SEQ?ID?NO：85)
GVFRVRPWYQSTSQS(SEQ?ID?NO：86)
VFRVRPWYQSTSQSG(SEQ?ID?NO：87)
CVFRVRPWYQSTSQS(SEQ?ID?NO：88)
VFRVRPWYQSTSQSC(SEQ?ID?NO：89)
Or its part.
The targeting peptide
In other embodiments, target peptide is the defined targeting peptides of preceding text.
According to the present invention, to, measure the aminoacid sequence of targeting peptide and it is optimized to be used for puting together the dsRNA that is used for sending with peptide.According to the present invention the useful peptide sequence of targeting peptide is described in the document.
Concerning each ligand family, different peptide sequence patterns are suitable.In one embodiment, can contain sequence x1-F-x2-YGG-x3 according to the present invention to the useful peptide of targeting LDL-receptor, wherein x1 and x3 are any aminoacid or comprise 2 to 40 amino acid whose fragments of peptides, and x2 is any aminoacid.Hussain, Strickland and Bakillah, Annu Rev Nutr.1999; 19:141-172.Hussain, Front Biosci.2001; 6:D417-D428.
According to useful targeting peptide of the present invention
Useful targeting peptide according to the present invention includes but not limited to the aminoacid sequence from arbitrary following part:
1. parathyroid hormone (PTH) and PTH GAP-associated protein GAP
P01270；PTHY_HUMAN
P22858；PTHR_MOUSE
Q811S6；Q811S6_MOUSE
P01270；PTHY_HUMAN
MIPAKDMAKV?MIVMLAICFL?TKSDGKSVKK?RSVSEIQLMH?NLGKHLNSME?RVEWLRKKLQ
DVHNFVALGA?PLAPRDAGSQ?RPRKKEDNVL?VESHEKSLGE?ADKADVNVLT?KAKSQ(SEQ?IDNO：90)
2. thyrotropin (TSH)
P01222；TSHB_HUMAN
P12656；TSHB_MOUSE
P01222；TSHB_HUMAN
MTALFLMSML?FGLACGQAMS?FCIPTEYTMH?IERRECAYCL?TINTTICAGY?CMTRDINGKL
FLPKYALSQD?VCTYRDFIYR?TVEIPGCPLH?VAPYFSYPVA?LSCKCGKCNT?DYSDCIHEAI
KTNYCTKPQK?SYLVGFSV(SEQ?ID?NO：91)
3.TSH releasing hormone
B2R8R1；B2R8R1_HUMAN
B2R8R1；B2R8R1_HUMAN
MPGPWLLLAL?ALTLNLTGVP?GGRAQPEAAQ?QEAVTAAEHP?GLDDFLRQVE?RLLFLRENIQ
RLQGDQGEHS?ASQIFQSDWL?SKRQHPGKRE?EEEEEGVEEE?EEEEGGAVGP?HKRQHPGRRE
DEASWSVDVT?QHKRQHPGRR?SPWLAYAVPK?RQHPGRRLAD?PKAQRSWEEE?EEEEEREEDL
MPEKRQHPGK?RALGGPCGPQ?GAYGQAGLLL?GLLDDLSRSQ?GAEEKRQHPG?RRAAWVREPL?EE(SEQ?ID?NO：92)
4.FSH/LH releasing hormone
P01148；GON1_HUMAN
O43555；GON2_HUMAN
P13562；GON1_MOUSE
P01148；GON1_HUMAN
MKPIQKLLAG?LILLTWCVEG?CSSQHWSYGL?RPGGKRDAEN?LIDSFQEIVK
EVGQLAETQR?FECTTHQPRS?PLRDLKGALE?SLIEEETGQK?KI(SEQ?ID?NO：93)
O43555；GON2_HUMAN
MASSRRGLLL?LLLLTAHLGP?SEAQHWSHGW?YPGGKRALSS?AQDPQNALRP
PGRALDTAAG?SPVQTAHGLP?SDALAPLDDS?MPWEGRTTAQ?WSLHRKRHLA
RTLLTAAREP?RPAPPSSNKV(SEQ?ID?NO：94)
5. corticotropin releasing hormone (CRH)
P06850；CRF_HUMAN
Q8CIT0；CRF_MOUSE
P06850；CRF_HUMAN
MRLPLLVSAG?VLLVALLPCP?PCRALLSRGP?VPGARQAPQH?PQPLDFFQPP?PQSEQPQQPQ
ARPVLLRMGE?EYFLRLGNLN?KSPAAPLSPA?SSLLAGGSGS?RPSPEQATAN?FFRVLLQQLL
LPRRSLDSPA?ALAERGARNA?LGGHQEAPER?ERRSEEPPIS?LDLTFHLLRE?VLEMARAEQL
AQQAHSNRKL?MEIIGK
(SEQ?ID?NO：95)
6. thyroliberin (ACTH)
P01189；COLI_HUMAN
P01193；COLI_MOUSE
P01189；COLI_HUMAN
MPRSCCSRSG?ALLLALLLQA?SMEVRGWCLE?SSQCQDLTTE?SNLLECIRAC
KPDLSAETPM?FPGNGDEQPL?TENPRKYVMG?HFRWDRFGRR?NSSSSGSSGA
GQKREDVSAG?EDCGPLPEGG?PEPRSDGAKP?GPREGKRSYS?MEHFRWGKPV
GKKRRPVKVY?PNGAEDESAE?AFPLEFKREL?TGQRLREGDG?PDGPADDGAG
AQADLEHSLL?VAAEKKDEGP?YRMEHFRWGS?PPKDKRYGGF?MTSEKSQTPL
VTLFKNAIIK?NAYKKGE(SEQ?ID?NO：96)
7. proteinase activated receptors (PAR) part and thrombin receptor agonist
P00734；THRB_HUMAN
P19221；THRB_MOUSE
P00734；THRB_HUMAN
MAHVRGLQLP?GCLALAALCS?LVHSQHVFLA?PQQARSLLQR?VRRANTFLEE
VRKGNLEREC?VEETCSYEEA?FEALESSTAT?DVFWAKYTAC?ETARTPRDKL
AACLEGNCAE?GLGTNYRGHV?NITRSGIECQ?LWRSRYPHKP?EINSTTHPGA
DLQENFCRNP?DSSTTGPWCY?TTDPTVRRQE?CSIPVCGQDQ?VTVAMTPRSE
GSSVNLSPPL?EQCVPDRGQQ?YQGRLAVTTH?GLPCLAWASA?QAKALSKHQD
FNSAVQLVEN?FCRNPDGDEE?GVWCYVAGKP?GDFGYCDLNY?CEEAVEEETG
DGLDEDSDRA?IEGRTATSEY?QTFFNPRTFG?SGEADCGLRP?LFEKKSLEDK
TERELLESYI?DGRIVEGSDA?EIGMSPWQVM?LFRKSPQELL?CGASLISDRW
VLTAAHCLLY?PPWDKNFTEN?DLLVRIGKHS?RTRYERNIEK?ISMLEKIYIH
PRYNWRENLD?RDIALMKLKK?PVAFSDYIHP?VCLPDRETAA?SLLQAGYKGR
VTGWGNLKET?WTANVGKGQP?SVLQVVNLPI?VERPVCKDST?RIRITDNMFC
AGYKPDEGKR?GDACEGDSGG?PFVMKSPFNN?RWYQMGIVSW?GEGCDRDGKY
GFYTHVFRLK?KWIQKVIDQF?GE(SEQ?ID?NO：97)
8. complement receptors part
P01024；CO3_HUMAN
P01027；CO3_MOUSE
P01024；CO3_HUMAN
MGPTSGPSLL?LLLLTHLPLA?LGSPMYSIIT?PNILRLESEE?TMVLEAHDAQ
GDVPVTVTVH?DFPGKKLVLS?SEKTVLTPAT?NHMGNVTFTI?PANREFKSEK
GRNKFVTVQA?TFGTQVVEKV?VLVSLQSGYL?FIQTDKTIYT?PGSTVLYRIF
TVNHKLLPVG?RTVMVNIENP?EGIPVKQDSL?SSQNQLGVLP?LSWDIPELVN
MGQWKIRAYY?ENSPQQVFST?EFEVKEYVLP?SFEVIVEPTE?KFYYIYNEKG
LEVTITARFL?YGKKVEGTAF?VIFGIQDGEQ?RISLPESLKR?IPIEDGSGEV
VLSRKVLLDG?VQNPRAEDLV?GKSLYVSATV?ILHSGSDMVQ?AERSGIPIVT
SPYQIHFTKT?PKYFKPGMPF?DLMVFVTNPD?GSPAYRVPVA?VQGEDTVQSL
TQGDGVAKLS?INTHPSQKPL?SITVRTKKQE?LSEAEQATRT?MQALPYSTVG
NSNNYLHLSV?LRTELRPGET?LNVNFLLRMD?RAHEAKIRYY?TYLIMNKGRL
LKAGRQVREP?GQDLVVLPLS?ITTDFIPSFR?LVAYYTLIGA?SGQREVVADS
VWVDVKDSCV?GSLVVKSGQS?EDRQPVPGQQ?MTLKIEGDHG?ARVVLVAVDK
GVFVLNKKNK?LTQSKIWDVV?EKADIGCTPG?SGKDYAGVFS?DAGLTFTSSS
GQQTAQRAEL?QCPQPAARRR?RSVQLTEKRM?DKVGKYPKEL?RKCCEDGMRE
NPMRFSCQRR?TRFISLGEAC?KKVFLDCCNY?ITELRRQHAR?ASHLGLARSN
LDEDIIAEEN?IVSRSEFPES?WLWNVEDLKE?PPKNGISTKL?MNIFLKDSIT
TWEILAVSMS?DKKGICVADP?FEVTVMQDFF?IDLRLPYSVV?RNEQVEIRAV
LYNYRQNQEL?KVRVELLHNP?AFCSLATTKR?RHQQTVTIPP?KSSLSVPYVI
VPLKTGLQEV?EVKAAVYHHF?ISDGVRKSLK?VVPEGIRMNK?TVAVRTLDPE
RLGREGVQKE?DIPPADLSDQ?VPDTESETRI?LLQGTPVAQM?TEDAVDAERL
KHLIVTPSGC?GEQNMIGMTP?TVIAVHYLDE?TEQWEKFGLE?KRQGALELIK
KGYTQQLAFR?QPSSAFAAFV?KRAPSTWLTA?YVVKVFSLAV?NLIAIDSQVL
CGAVKWLILE?KQKPDGVFQE?DAPVIHQEMI?GGLRNNNEKD?MALTAFVLIS
LQEAKDICEE?QVNSLPGSIT?KAGDFLEANY?MNLQRSYTVA?IAGYALAQMG
RLKGPLLNKF?LTTAKDKNRW?EDPGKQLYNV?EATSYALLAL?LQLKDFDFVP
PVVRWLNEQR?YYGGGYGSTQ?ATFMVFQALA?QYQKDAPDHQ?ELNLDVSLQL
PSRSSKITHR?IHWESASLLR?SEETKENEGF?TVTAEGKGQG?TLSVVTMYHA
KAKDQLTCNK?FDLKVTIKPA?PETEKRPQDA?KNTMILEICT?RYRGDQDATM
SILDISMMTG?FAPDTDDLKQ?LANGVDRYIS?KYELDKAFSD?RNTLIIYLDK
VSHSEDDCLA?FKVHQYFNVE?LIQPGAVKVY?AYYNLEESCT?RFYHPEKEDG
KLNKLCRDEL?CRCAEENCFI?QKSDDKVTLE?ERLDKACEPG?VDYVYKTRLV
KVQLSNDFDE?YIMAIEQTIK?SGSDEVQVGQ?QRTFISPIKC?REALKLEEKK
HYLMWGLSSD?FWGEKPNLSY?IIGKDTWVEH?WPEEDECQDE?ENQKQCQDLG
AFTESMVVFG?CPN(SEQ?ID?NO：98)
P0C0L5；CO4B_HUMAN
MRLLWGLIWA?SSFFTLSLQK?PRLLLFSPSV?VHLGVPLSVG?VQLQDVPRGQ?VVKGSVFLRN
PSRNNVPCSP?KVDFTLSSER?DFALLSLQVP?LKDAKSCGLH?QLLRGPEVQL?VAHSPWLKDS
LSRTTNIQGI?NLLFSSRRGH?LFLQTDQPIY?NPGQRVRYRV?FALDQKMRPS?TDTITVMVEN
SHGLRVRKKE?VYMPSSIFQD?DFVIPDISEP?GTWKISARFS?DGLESNSSTQ?FEVKKYVLPN
FEVKITPGKP?YILTVPGHLD?EMQLDIQARY?IYGKPVQGVA?YVRFGLLDED?GKKTFFRGLE
SQTKLVNGQS?HISLSKAEFQ?DALEKLNMGI?TDLQGLRLYV?AAAIIESPGG?EMEEAELTSW
YFVSSPFSLD?LSKTKRHLVP?GAPFLLQALV?REMSGSPASG?IPVKVSATVS?SPGSVPEVQD
IQQNTDGSGQ?VSIPIIIPQT?ISELQLSVSA?GSPHPAIARL?TVAAPPSGGP?GFLSIERPDS
RPPRVGDTLN?LNLRAVGSGA?TFSHYYYMIL?SRGQIVFMNR?EPKRTLTSVS?VFVDHHLAPS
FYFVAFYYHG?DHPVANSLRV?DVQAGACEGK?LELSVDGAKQ?YRNGESVKLH?LETDSLALVA
LGALDTALYA?AGSKSHKPLN?MGKVFEAMNS?YDLGCGPGGG?DSALQVFQAA?GLAFSDGDQW
TLSRKRLSCP?KEKTTRKKRN?VNFQKAINEK?LGQYASPTAK?RCCQDGVTRL?PMMRSCEQRA
ARVQQPDCRE?PFLSCCQFAE?SLRKKSRDKG?QAGLQRALEI?LQEEDLIDED?DIPVRSFFPE
NWLWRVETVD?RFQILTLWLP?DSLTTWEIHG?LSLSKTKGLC?VATPVQLRVF?REFHLHLRLP
MSVRRFEQLE?LRPVLYNYLD?KNLTVSVHVS?PVEGLCLAGG?GGLAQQVLVP?AGSARPVAFS
VVPTAAAAVS?LKVVARGSFE?FPVGDAVSKV?LQIEKEGAIH?REELVYELNP?LDHRGRTLEI
PGNSDPNMIP?DGDFNSYVRV?TASDPLDTLG?SEGALSPGGV?ASLLRLPRGC?GEQTMIYLAP
TLAASRYLDK?TEQWSTLPPE?TKDHAVDLIQ?KGYMRIQQFR?KADGSYAAWL?SRDSSTWLTA
FVLKVLSLAQ?EQVGGSPEKL?QETSNWLLSQ?QQADGSFQDL?SPVIHRSMQG?GLVGNDETVA
LTAFVTIALH?HGLAVFQDEG?AEPLKQRVEA?SISKANSFLG?EKASAGLLGA?HAAAITAYAL
SLTKAPVDLL?GVAHNNLMAM?AQETGDNLYW?GSVTGSQSNA?VSPTPAPRNP?SDPMPQAPAL
WIETTAYALL?HLLLHEGKAE?MADQASAWLT?RQGSFQGGFR?STQDTVIALD?ALSAYWIASH
TTEERGLNVT?LSSTGRNGFK?SHALQLNNRQ?IRGLEEELQF?SLGSKINVKV?GGNSKGTLKV
LRTYNVLDMK?NTTCQDLQIE?VTVKGHVEYT?MEANEDYEDY?EYDELPAKDD?PDAPLQPVTP
LQLFEGRRNR?RRREAPKVVE?EQESRVHYTV?CIWRNGKVGL?SGMAIADVTL?LSGFHALRAD
LEKLTSLSDR?YVSHFETEGP?HVLLYFDSVP?TSRECVGFEA?VQEVPVGLVQ?PASATLYDYY
NPERRCSVFY?GAPSKSRLLA?TLCSAEVCQC?AEGKCPRQRR?ALERGLQDED?GYRMKFACYY
PRVEYGFQVK?VLREDSRAAF?RLFETKITQV?LHFTKDVKAA?ANQMRNFLVR?ASCRLRLEPG
KEYLIMGLDG?ATYDLEGHPQ?YLLDSNSWIE?EMPSERLCRS?TRQRAACAQL?NDFLQEYGTQ
GCQV
(SEQ?ID?NO：99)
9.LDL the part of receptor family
P05067；A4_HUMAN
P12023；A4_MOUSE
P05067；A4_HUMAN
MLPGLALLLL?AAWTARALEV?PTDGNAGLLA?EPQIAMFCGR?LNMHMNVQNG?KWDSDPSGTK
TCIDTKEGIL?QYCQEVYPEL?QITNVVEANQ?PVTIQNWCKR?GRKQCKTHPH?FVIPYRCLVG
EFVSDALLVP?DKCKFLHQER?MDVCETHLHW?HTVAKETCSE?KSTNLHDYGM?LLPCGIDKFR
GVEFVCCPLA?EESDNVDSAD?AEEDDSDVWW?GGADTDYADG?SEDKVVEVAE?EEEVAEVEEE
EADDDEDDED?GDEVEEEAEE?PYEEATERTT?SIATTTTTTT?ESVEEVVREV?CSEQAETGPC
RAMISRWYFD?VTEGKCAPFF?YGGCGGNRNN?FDTEEYCMAV?CGSAMSQSLL?KTTQEPLARD
PVKLPTTAAS?TPDAVDKYLE?TPGDENEHAH?FQKAKERLEA?KHRERMSQVM?REWEEAERQA
KNLPKADKKA?VIQHFQEKVE?SLEQEAANER?QQLVETHMAR?VEAMLNDRRR?LALENYITAL
QAVPPRPRHV?FNMLKKYVRA?EQKDRQHTLK?HFEHVRMVDP?KKAAQIRSQV?MTHLRVIYER
MNQSLSLLYN?VPAVAEEIQD?EVDELLQKEQ?NYSDDVLANM?ISEPRISYGN?DALMPSLTET
KTTVELLPVN?GEFSLDDLQP?WHSFGADSVP?ANTENEVEPV?DARPAADRGL?TTRPGSGLTN
IKTEEISEVK?MDAEFRHDSG?YEVHHQKLVF?FAEDVGSNKG?AIIGLMVGGV?VIATVIVITL
VMLKKKQYTS?IHHGVVEVDA?AVTPEERHLS?KMQQNGYENP?TYKFFEQMQN
(SEQ?ID?NO：100)
10. endocrine and external secretion receptors ligand
P01241；SOMA_HUMAN
P06880；SOMA_MOUSE
Q9UBU3；GHRL_HUMAN
Q9EQX0；GHRL_MOUSE
P01241；SOMA_HUMAN
MATGSRTSLL?LAFGLLCLPW?LQEGSAFPTI?PLSRLFDNAM?LRAHRLHQLA?FDTYQEFEEA
YIPKEQKYSF?LQNPQTSLCF?SESIPTPSNR?EETQQKSNLE?LLRISLLLIQ?SWLEPVQFLR
SVFANSLVYG?ASDSNVYDLL?KDLEEGIQTL?MGRLEDGSPR?TGQIFKQTYS?KFDTNSHNDD
ALLKNYGLLY?CFRKDMDKVE?TFLRIVQCRS?VEGSCGF
(SEQ?ID?NO：101)
Q9UBU3；GHRL_HUMAN
MPSPGTVCSL?LLLGMLWLDL?AMAGSSFLSP?EHQRVQQRKE?SKKPPAKLQP?RALAGWLRPE
DGGQAEGAED?ELEVRFNAPF?DVGIKLSGVQ?YQQHSQALGK?FLQDILWEEA?KEAPADK(SEQ?ID?NO：102)
11. transforming growth factor part
P01137；TGFB1_HUMAN
P04202；TGFB1_MOUSE
P01137；TGFB1_HUMAN
MPPSGLRLLL?LLLPLLWLLV?LTPGRPAAGL?STCKTIDMEL?VKRKRIEAIR?GQILSKLRLA
SPPSQGEVPP?GPLPEAVLAL?YNSTRDRVAG?ESAEPEPEPE?ADYYAKEVTR?VLMVETHNEI
YDKFKQSTHS?IYMFFNTSEL?REAVPEPVLL?SRAELRLLRL?KLKVEQHVEL?YQKYSNNSWR
YLSNRLLAPS?DSPEWLSFDV?TGVVRQWLSR?GGEIEGFRLS?AHCSCDSRDN?TLQVDINGFT
TGRRGDLATI?HGMNRPFLLL?MATPLERAQH?LQSSRHRRAL?DTNYCFSSTE?KNCCVRQLYI
DFRKDLGWKW?IHEPKGYHAN?FCLGPCPYIW?SLDTQYSKVL?ALYNQHNPGA?SAAPCCVPQA
LEPLPIVYYV?GRKPKVEQLS?NMIVRSCKCS
(SEQ?ID?NO：103)
12. chemokine receptor ligands
P13500；CCL2_HUMAN
P10148；CCL2_MOUSE
P13500；CCL2_HUMAN
MKVSAALLCL?LLIAATFIPQ?GLAQPDAINA?PVTCCYNFTN?RKISVQRLAS?YRRITSSKCP
KEAVIFKTIV?AKEICADPKQ?KWVQDSMDHL?DKQTQTPKT(SEQ?ID?NO：104)
13. integrin
P05556；ITB1_HUMAN
P09055；ITB1_MOUSE
P05556；ITB1_HUMAN
MNLQPIFWIG?LISSVCCVFA?QTDENRCLKA?NAKSCGECIQ?AGPNCGWCTN?STFLQEGMPT
SARCDDLEAL?KKKGCPPDDI?ENPRGSKDIK?KNKNVTNRSK?GTAEKLKPED?ITQIQPQQLV
LRLRSGEPQT?FTLKFKRAED?YPIDLYYLMD?LSYSMKDDLE?NVKSLGTDLM?NEMRRITSDF
RIGFGSFVEK?TVMPYISTTP?AKLRNPCTSE?QNCTSPFSYK?NVLSLTNKGE?VFNELVGKQR
ISGNLDSPEG?GFDAIMQVAV?CGSLIGWRNV?TRLLVFSTDA?GFHFAGDGKL?GGIVLPNDGQ
CHLENNMYTM?SHYYDYPSIA?HLVQKLSENN?IQTIFAVTEE?FQPVYKELKN?LIPKSAVGTL
SANSSNVIQL?IIDAYNSLSS?EVILENGKLS?EGVTISYKSY?CKNGVNGTGE?NGRKCSNISI
GDEVQFEISI?TSNKCPKKDS?DSFKIRPLGF?TEEVEVILQY?ICECECQSEG?IPESPKCHEG
NGTFECGACR?CNEGRVGRHC?ECSTDEVNSE?DMDAYCRKEN?SSEICSNNGE?CVCGQCVCRK
RDNTNEIYSG?KFCECDNFNC?DRSNGLICGG?NGVCKCRVCE?CNPNYTGSAC?DCSLDTSTCE
ASNGQICNGR?GICECGVCKC?TDPKFQGQTC?EMCQTCLGVC?AEHKECVQCR?AFNKGEKKDT
CTQECSYFNI?TKVESRDKLP?QPVQPDPVSH?CKEKDVDDCW?FYFTYSVNGN?NEVMVHVVEN
PECPTGPDII?PIVAGVVAGI?VLIGLALLLI?WKLLMIIHDR?REFAKFEKEK?MNAKWDTGEN
PIYKSAVTTV?VNPKYEGK(SEQ?ID?NO：105)
14. interleukin
Q13169；Q13169_HUMAN
Q0PGS4；Q0PGS4_MOUSE
Q13169；Q13169_HUMAN
MYRMQLLSCI?ALILALVTNS?APTSSSTKKT?KKTQLQLEHL?LLDLQMILNG?INNYKNPKLT
RMLTFKFYMP?KKATELKQLQ?CLEEELKPLE?EVLNLAQSKN?FHLRPRDLIS?NINVIVLELK
GSETTFMCEY?ADETATIVEF?LNRWITFCQS?IISTLT
(SEQ?ID?NO：106)
15. differentiation factor appearance bone differentiation factor
P13497；BMP1_HUMAN
P98063；BMP1_MOUSE
P13497；BMP1_HUMAN
MPGVARLPLL?LGLLLLPRPG?RPLDLADYTY?DLAEEDDSEP?LNYKDPCKAA?AFLGDIALDE
EDLRAFQVQQ?AVDLRRHTAR?KSSIKAAVPG?NTSTPSCQST?NGQPQRGACG?RWRGRSRSRR
AATSRPERVW?PDGVIPFVIG?GNFTGSQRAV?FRQAMRHWEK?HTCVTFLERT?DEDSYIVFTY
RPCGCCSYVG?RRGGGPQAIS?IGKNCDKFGI?VVHELGHVVG?FWHEHTRPDR?DRHVSIVREN
IQPGQEYNFL?KMEPQEVESL?GETYDFDSIM?HYARNTFSRG?IFLDTIVPKY?EVNGVKPPIG
QRTRLSKGDI?AQARKLYKCP?ACGETLQDST?GNFSSPEYPN?GYSAHMHCVW?RISVTPGEKI
ILNFTSLDLY?RSRLCWYDYV?EVRDGFWRKA?PLRGRFCGSK?LPEPIVSTDS?RLWVEFRSSS
NWVGKGFFAV?YEAICGGDVK?KDYGHIQSPN?YPDDYRPSKV?CIWRIQVSEG?FHVGLTFQSF
EIERHDSCAY?DYLEVRDGHS?ESSTLIGRYC?GYEKPDDIKS?TSSRLWLKFV?SDGSINKAGF
AVNFFKEVDE?CSRPNRGGCE?QRCLNTLGSY?KCSCDPGYEL?APDKRRCEAA?CGGFLTKLNG
SITSPGWPKE?YPPNKNCIWQ?LVAPTQYRIS?LQFDFFETEG?NDVCKYDFVE?VRSGLTADSK
LHGKFCGSEK?PEVITSQYNN?MRVEFKSDNT?VSKKGFKAHF?FSDKDECSKD?NGGCQQDCVN
TFGSYECQCR?SGFVLHDNKH?DCKEAGCDHK?VTSTSGTITS?PNWPDKYPSK?KECTWAISST
PGHRVKLTFM?EMDIESQPEC?AYDHLEVFDG?RDAKAPVLGR?FCGSKKPEPV?LATGSRMFLR
FYSDNSVQRK?GFQASHATEC?GGQVRADVKT?KDLYSHAQFG?DNNYPGGVDC?EWVIVAEEGY
GVELVFQTFE?VEEETDCGYD?YMELFDGYDS?TAPRLGRYCG?SGPPEEVYSA?GDSVLVKFHS
DDTITKKGFH?LRYTSTKFQD?TLHSRK(SEQ?ID?NO：107)
16. gastrin releasing peptide
P07492；GRP_HUMAN
Q8R1I2；GRP_MOUSE
P07492；GRP_HUMAN
MRGSELPLVL?LALVLCLAPR?GRAVPLPAGG?GTVLTKMYPR?GNHWAVGHLM?GKKSTGESSS
VSERGSLKQQ?LREYIRWEEA?ARNLLGLIEA?KENRNHQPPQ?PKALGNQQPS?WDSEDSSNFK
DVGSKGKVGR?LSAPGSQREG?RNPQLNQQ(SEQ?ID?NO：108)
17. vasoactive intestinal peptide (VIP)
P01282；VIP_HUMAN
P32648；VIP_MOUSE
P01282；VIP_HUMAN
MDTRNKAQLL?VLLTLLSVLF?SQTSAWPLYR?APSALRLGDR?IPFEGANEPD?QVSLKEDIDM
LQNALAENDT?PYYDVSRNAR?HADGVFTSDF?SKLLGQLSAK?KYLESLMGKR?VSSNISEDPV
PVKRHSDAVF?TDNYTRLRKQ?MAVKKYLNSI?LNGKRSSEGE?SPDFPEELEK(SEQ?ID?NO：109)
18. insulin and insulin like growth factor
P01308；INS_HUMAN
P01343；IGF1A_HUMAN
P05019；IGF1B_HUMAN
P05017；IGF1_MOUSE
P01308；INS_HUMAN
MALWMRLLPL?LALLALWGPD?PAAAFVNQHL?CGSHLVEALY?LVCGERGFFY?TPKTRREAED
LQVGQVELGG?GPGAGSLQPL?ALEGSLQKRG?IVEQCCTSIC?SLYQLENYCN(SEQ?ID?NO：110)
P01343；IGF1A_HUMAN
MGKISSLPTQ?LFKCCFCDFL?KVKMHTMSSS?HLFYLALCLL?TFTSSATAGP?ETLCGAELVD
ALQFVCGDRG?FYFNKPTGYG?SSSRRAPQTG?IVDECCFRSC?DLRRLEMYCA?PLKPAKSARS
VRAQRHTDMP?KTQKEVHLKN?ASRGSAGNKN?YRM(SEQ?ID?NO：111)
P05019；IGF1B_HUMAN
MGKISSLPTQ?LFKCCFCDFL?KVKMHTMSSS?HLFYLALCLL?TFTSSATAGP?ETLCGAELVD
ALQFVCGDRG?FYFNKPTGYG?SSSRRAPQTG?IVDECCFRSC?DLRRLEMYCA?PLKPAKSARS
VRAQRHTDMP?KTQKYQPPST?NKNTKSQRRK?GWPKTHPGGE?QKEGTEASLQ?IRGKKKEQRR
EIGSRNAECR?GKKGK
(SEQ?ID?NO：112)
19. calcitonin and calcitonin gene related peptide
P01258；CALC_HUMAN
P70160；CALC_MOUSE
P01258；CALC_HUMAN
MGFQKFSPFL?ALSILVLLQA?GSLHAAPFRS?ALESSPADPA?TLSEDEARLL?LAALVQDYVQ
MKASELEQEQ?EREGSSLDSP?RSKRCGNLST?CMLGTYTQDF?NKFHTFPQTA?IGVGAPGKKR
DMSSDLERDH?RPHVSMPQNA?N(SEQ?ID?NO：113)
20. the part of inflammatory cell appearance mastocyte, eosinocyte, macrophage, mononuclear cell and neutrophil
P09603；CSF1_HUMAN
P07141；CSF1_MOUSE
P0C0L5；CO4B_HUMAN
P01029；CO4B_MOUSE
P09603；CSF1_HUMAN
MTAPGAAGRC?PPTTWLGSLL?LLVCLLASRS?ITEEVSEYCS?HMIGSGHLQS?LQRLIDSQME
TSCQITFEFV?DQEQLKDPVC?YLKKAFLLVQ?DIMEDTMRFR?DNTPNAIAIV?QLQELSLRLK
SCFTKDYEEH?DKACVRTFYE?TPLQLLEKVK?NVFNETKNLL?DKDWNIFSKN?CNNSFAECSS
QDVVTKPDCN?CLYPKAIPSS?DPASVSPHQP?LAPSMAPVAG?LTWEDSEGTE?GSSLLPGEQP
LHTVDPGSAK?QRPPRSTCQS?FEPPETPVVK?DSTIGGSPQP?RPSVGAFNPG?MEDILDSAMG
TNWVPEEASG?EASEIPVPQG?TELSPSRPGG?GSMQTEPARP?SNFLSASSPL?PASAKGQQPA
DVTGTALPRV?GPVRPTGQDW?NHTPQKTDHP?SALLRDPPEP?GSPRISSLRP?QGLSNPSTLS
AQPQLSRSHS?SGSVLPLGEL?EGRRSTRDRR?SPAEPEGGPA?SEGAARPLPR?FNSVPLTDTG
HERQSEGSSS?PQLQESVFHL?LVPSVILVLL?AVGGLLFYRW?RRRSHQEPQR?ADSPLEQPEG
SPLTQDDRQV?ELPV(SEQ?ID?NO：114)
Useful other targeting peptide according to the present invention includes but not limited to following:
GTFVYGGCRAKRNNFKSAED(SEQ?ID?NO：115)
GPFFYGGCGGNRNNFDTEEY(SEQ?ID?NO：116)
GTFFYGGCRGKRNNFKTEEY(SEQ?ID?NO：117)
GTFFYGGSRGKRNNFKTEEY(SEQ?ID?NO：118)
GRFFYGGSRGKRNNFRTEEY(SEQ?ID?NO：119)
GTFFYGGSRGRRNNFRTEEY(SEQ?ID?NO：120)
CTFVYGGCRAKRNNFKSAED(SEQ?ID?NO：121)
CPFFYGGCGGNRNNFDTEEY(SEQ?ID?NO：122)
CTFFYGGCRGKRNNFKTEEY(SEQ?ID?NO：123)
CTFFYGGSRGKRNNFKTEEY(SEQ?ID?NO：124)
CRFFYGGSRGKRNNFRTEEY(SEQ?ID?NO：125)
CTFFYGGSRGRRNNFRTEEY(SEQ?ID?NO：126)
TFVYGGCRAKRNNFKSAEDG(SEQ?ID?NO：127)
PFFYGGCGGNRNNFDTEEYG(SEQ?ID?NO：128)
TFFYGGCRGKRNNFKTEEYG(SEQ?ID?NO：129)
TFFYGGSRGKRNNFKTEEYG(SEQ?ID?NO：130)
RFFYGGSRGKRNNFRTEEYG(SEQ?ID?NO：131)
TFFYGGSRGRRNNFRTEEYG(SEQ?ID?NO：132)
TFVYGGCRAKRNNFKSAEDC(SEQ?ID?NO：133)
PFFYGGCGGNRNNFDTEEYC(SEQ?ID?NO：134)
TFFYGGCRGKRNNFKTEEYC(SEQ?ID?NO：135)
TFFYGGSRGKRNNFKTEEYC(SEQ?ID?NO：136)
RFFYGGSRGKRNNFRTEEYC(SEQ?ID?NO：137)
TFFYGGSRGRRNNFRTEEYC(SEQ?ID?NO：138)
STEELRVRLASHLRKLRKRLL(SEQ?ID?NO：149)
SSVIDALQYKLEGTTRLTRKRGLKLATALSLSNKFVEGS(SEQ?ID?NO：150)
EELRVRLASHLRKLRKRLLRDADDLQK(SEQ?ID?NO：154)
GQSTEELRARLASHLRKLRKR(SEQ?ID?NO：155)
RLASHLRKLRKRLLRD(SEQ?ID?NO：156)
H2N-c[D(Cys-Ser-Lys-Cys)]Gly-Peg12-Lys(SEQ?ID?NO：151)
H2N-c[Cys-Phe-Thr-Lys-D-Trp-Phe-Phe-Cys]-Peg12-Lys(SEQ?ID?NO：152)
H2N-Thr-Phe-Thr-Lys-D-Trp-Phe-Phe-D-Phe-Peg12-Lys(SEQ?ID?NO：153)
In one embodiment, peptide of the present invention and transposition structural domain (for example, the transposition structural domain of neurotoxin) are puted together.Useful neurotoxin transposition structural domain peptide sequence according to the present invention includes but not limited to following.Peptide sequence is selected from any subunit in the sequence.Based on the sequence that meets the present invention's regulation, select fragments of peptides.
1.A type botulic neurotoxin (BoNT/A) (EC 3.4.24.69) (Bontoxilysin-A)
P10845；BXA1_CLOBO
MPFVNKQFNY?KDPVNGVDIA?YIKIPNVGQM?QP1VKAFKIHN?KIWVIPERDT?FTNPEEGDLN
PPPEAKQVPV?SYYDSTYLST?DNEKDNYLKG?VTKLFERIYS?TDLGRMLLTS?IVRGIPFWGG
STIDTELKVI?DTNCINVIQP?DGSYRSEELN?LVIIGPSADI?IQFECKSFGH?EVLNLTRNGY
GSTQYIRFSP?DFTFGFEESL?EVDTNPLLGA?GKFATDPAVT?LAHELIHAGH?RLYGIAINPN
RVFKVNTNAY?YEMSGLEVSF?EELRTFGGHD?AKFIDSLQEN?EFRLYYYNKF?KDIASTLNKA
KSIVGTTASL?QYMKNVFKEK?YLLSEDTSGK?FSVDKLKFDK?LYKMLTEIYT?EDNFVKFFKV
LNRKTYLNFD?KAVFKINIVP?KVNYTIYDGF?NLRNTNLAAN?FNGQNTEINN?MNFTKLKNFT
GLFEFYKLLC?VRGIITSKTK?SLDKGYNKAL?NDLCIKVNNW?DLFFSPSEDN?FTNDLNKGEE
ITSDTNIEAA?EENISLDLIQ?QYYLTFNFDN?EPENISIENL?SSDIIGQLEL?MPNIERFPNG
KKYELDKYTM?FHYLRAQEFE?HGKSRIALTN?SVNEALLNPS?RVYTFFSSDY?VKKVNKATEA
AMFLGWVEQL?VYDFTDETSE?VSTTDKIADI?TIIIPYIGPA?LNIGNMLYKD?DFVGALIFSG
AVILLEFIPE?IAIPVLGTFA?LVSYIANKVL?TVQTIDNALS?KRNEKWDEVY?KYIVTNWLAK
VNTQIDLIRK?KMKEALENQA?EATKAIINYQ?YNQYTEEEKN?NINFNIDDLS?SKLNESINKA
MININKFLNQ?CSVSYLMNSM?IPYGVKRLED?FDASLKDALL?KYIYDNRGTL?IGQVDRLKDK
VNNTLSTDIP?FQLSKYVDNQ?RLLSTFTEYI?KNIINTSILN?LRYESNHLID?LSRYASKINI
GSKVNFDPID?KNQIQLFNLE?SSKIEVILKN?AIVYNSMYEN?FSTSFWIRIP?KYFNSISLNN
EYTIINCMEN?NSGWKVSLNY?GEIIWTLQDT?QEIKQRVVFK?YSQMINISDY?INRWIFVTIT
NNRLNNSKIY?INGRLIDQKP?ISNLGNIHAS?NNIMFKLDGC?RDTHRYIWIK?YFNLFDKELN
EKEIKDLYDN?QSNSGILKDF?WGDYLQYDKP?YYMLNLYDPN?KYVDVNNVGI?RGYMYLKGPR
GSVMTTNIYL?NSSLYRGTKF?IIKKYASGNK?DNIVRNNDRV?YINVVVKNKE?YRLATNASQA
GVEKILSALE?IPDVGNLSQV?VVMKSKNDQG?ITNKCKMNLQ?DNNGNDIGFI?GFHQFNNIAK
LVASNWYNRQ?IERSSRTLGC?SWEFIPVDDG?WGERPL(SEQ?ID?NO：139)
2.B type botulic neurotoxin (BoNT/B) (EC 3.4.24.69) (Bontoxilysin-B)
B1INP5；BXB_CLOBK
MPVTINNFNY?NDPIDNNNII?MMEPPFARGT?GRYYKAFKIT?DRIWIIPERY?TFGYKPEDFN
KSSGIFNRDV?CEYYDPDYLN?TNDKKNIFLQ?TMIKLFNRIK?SKPLGEKLLE?MIINGIPYLG
DRRVPLEEFN?TNIASVTVNK?LISNPGEVER?KKGIFANLII?FGPGPVLNEN?ETIDIGIQNH
FASREGFGGI?MQMKFCPEYV?SVFNNVQENK?GASIFNRRGY?FSDPALILMH?ELIHVLHGLY
GIKVDDLPIV?PNEKKFFMQS?TDAIQAEELY?TFGGQDPSII?TPSTDKSIYD?KVLQNFRGIV
DRLNKVLVCI?SDPNININIY?KNKFKDKYKF?VEDSEGKYSI?DVESFDKLYK?SLMFGFTETN
IAENYKIKTR?ASYFSDSLPP?VKIKNLLDNE?IYTIEEGFNI?SDKDMEKEYR?GQNKAINKQA
YEEISKEHLA?VYKIQMCKSV?KAPGICIDVD?NEDLFFIADK?NSFSDDLSKN?ERIEYNTQSN
YIENDFPINE?LILDTDLISK?IELPSENTES?LTDFNVDVPV?YEKQPAIKKI?FTDENTIFQY
LYSQTFPLDI?RDISLTSSFD?DALLFSNKVY?SFFSMDYIKT?ANKVVEAGLF?AGWVKQIVND
FVIEANKSNT?MDKIADISLI?VPYIGLALNV?GNETAKGNFE?NAFEIAGASI?LLEFIPELLI
PVVGAFLLES?YIDNKNKIIK?TIDNALTKRN?EKWSDMYGLI?VAQWLSTVNT?QFYTIKEGMY
KALNYQAQAL?EEIIKYRYNI?YSEKEKSNIN?IDFNDINSKL?NEGINQAIDN?INNFINGCSV
SYLMKKMIPL?AVEKLLDFDN?TLKKNLLNYI?DENKLYLIGS?AEYEKSKVNK?YLKTIMPFDL
SIYTNDTILI?EMFNKYNSEI?LNNIILNLRY?KDNNLIDLSG?YGAKVEVYDG?VELNDKNQFK
LTSSANSKIR?VTQNQNIIFN?SVFLDFSVSF?WIRIPKYKND?GIQNYIHNEY?TIINCMKNNS
GWKISIRGNR?IIWTLIDING?KTKSVFFEYN?IREDISEYIN?RWFFVTITNN?LNNAKIYING
KLESNTDIKD?IREVIANGEI?IFKLDGDIDR?TQFIWMKYFS?IFNTELSQSN?IEERYKIQSY
SEYLKDFWGN?PLMYNKEYYM?FNAGNKNSYI?KLKKDSPVGE?ILTRSKYNQN?SKYINYRDLY
IGEKFIIRRK?SNSQSINDDI?VRKEDYIYLD?FFNLNQEWRV?YTYKYFKKEE?EKLFLAPISD
SDEFYNTIQI?KEYDEQPTYS?CQLLFKKDEE?STDEIGLIGI?HRFYESGIVF?EEYKDYFCIS
KWYLKEVKRK?PYNLKLGCNW?QFIPKDEGWT?E(SEQ?ID?NO：140)
3.C1 type botulic neurotoxin (BoNT/C1) (EC 3.4.24.69) (Bontoxilysin-C1)
P18640；BXC1_CLOBO
MPITINNFNY?SDPVDNKNIL?YLDTHLNTLA?NEPEKAFRIT?GNIWVIPDRF?SRNSNPNLNK
PPRVTSPKSG?YYDPNYLSTD?SDKDPFLKEI?IKLFKRINSR?EIGEELIYRL?STDIPFPGNN
NTPINTFDFD?VDFNSVDVKT?RQGNNWVKTG?SINPSVIITG?PRENIIDPET?STFKLTNNTF
AAQEGFGALS?IISISPRFML?TYSNATNDVG?EGRFSKSEFC?MDPILILMHE?LNHAMHNLYG
IAIPNDQTIS?SVTSNIFYSQ?YNVKLEYAEI?YAFGGPTIDL?IPKSARKYFE?EKALDYYRSI
AKRLNSITTA?NPSSFNKYIG?EYKQKLIRKY?RFVVESSGEV?TVNRNKFVEL?YNELTQIFTE
FNYAKIYNVQ?NRKIYLSNVY?TPVTANILDD?NVYDIQNGFN?IPKSNLNVLF?MGQNLSRNPA
LRKVNPENML?YLFTKFCHKA?IDGRSLYNKT?LDCRELLVKN?TDLPFIGDIS?DVKTDIFLRK
DINEETEVIY?YPDNVSVDQV?ILSKNTSEHG?QLDLLYPSID?SESEILPGEN?QVFYDNRTQN
VDYLNSYYYL?ESQKLSDNVE?DFTFTRSIEE?ALDNSAKVYT?YFPTLANKVN?AGVQGGLFLM
WANDVVEDFT?TNILRKDTLD?KISDVSAIIP?YIGPALNISN?SVRRGNFTEA?FAVTGVTILL
EAFPEFTIPA?LGAFVIYSKV?QERNEIIKTI?DNCLEQRIKR?WKDSYEWMMG?TWLSRIITQF
NNISYQMYDS?LNYQAGAIKA?KIDLEYKKYS?GSDKENIKSQ?VENLKNSLDV?KISEAMNNIN
KFIRECSVTY?LFKNMLPKVI?DELNEFDRNT?KAKLINLIDS?HNIILVGEVD?KLKAKVNNSF
QNTIPFNIFS?YTNNSLLKDI?INEYFNNIND?SKILSLQNRK?NTLVDTSGYN?AEVSEEGDVQ
LNPIFPFDFK?LGSSGEDRGK?VIVTQNENIV?YNSMYESFSI?SFWIRINKWV?SNLPGYTIID
SVKNNSGWSI?GIISNFLVFT?LKQNEDSEQS?INFSYDISNN?APGYNKWFFV?TVTNNMMGNM
KIYINGKLID?TIKVKELTGI?NFSKTITFEI?NKIPDTGLIT?SDSDNINMWI?RDFYIFAKEL
DGKDINILFN?SLQYTNVVKD?YWGNDLRYNK?EYYMVNIDYL?NRYMYANSRQ?IVFNTRRNNN
DFNEGYKIII?KRIRGNTNDT?RVRGGDILYF?DMTINNKAYN?LFMKNETMYA?DNHSTEDIYA
IGLREQTKDI?NDNIIFQIQP?MNNTYYYASQ?IFKSNFNGEN?ISGICSIGTY?RFRLGGDWYR
HNYLVPTVKQ?GNYASLLEST?STHWGFVPVS?E(SEQ?ID?NO：141)
4.D type botulic neurotoxin (BoNT/D) (EC 3.4.24.69) (Bontoxilysin-D)
P19321；BXD_CLOBO
MTWPVKDFNY?SDPVNDNDIL?YLRIPQNKLI?TTPVKAFMIT?QNIWVIPERF?SSDTNPSLSK
PPRPTSKYQS?YYDPSYLSTD?EQKDTFLKGI?IKLFKRINER?DIGKKLINYL?VVGSPFMGDS
STPEDTFDFT?RHTTNIAVEK?FENGSWKVTN?IITPSVLIFG?PLPNILDYTA?SLTLQGQQSN
PSFEGFGTLS?ILKVAPEFLL?TFSDVTSNQS?SAVLGKSIFC?MDPVIALMHE?LTHSLHQLYG
INIPSDKRIR?PQVSEGFFSQ?DGPNVQFEEL?YTFGGLDVEI?IPQIERSQLR?EKALGHYKDI
AKRLNNINKT?IPSSWISNID?KYKKIFSEKY?NFDKDNTGNF?VVNIDKFNSL?YSDLTNVMSE
VVYSSQYNVK?NRTHYFSRHY?LPVFANILDD?NIYTIRDGFN?LTNKGFNIEN?SGQNIERNPA
LQKLSSESVV?DLFTKVCLRL?TKNSRDDSTC?IKVKNNRLPY?VADKDSISQE?IFENKIITDE
TNVQNYSDKF?SLDESILDGQ?VPINPEIVDP?LLPNVNMEPL?NLPGEEIVFY?DDITKYVDYL
NSYYYLESQK?LSNNVENITL?TTSVEEALGY?SNKIYTFLPS?LAEKVNKGVQ?AGLFLNWANE
VVEDFTTNIM?KKDTLDKISD?VSVIIPYIGP?ALNIGNSALR?GNFNQAFATA?GVAFLLEGFP
EFTIPALGVF?TFYSSIQERE?KIIKTIENCL?EQRVKRWKDS?YQWMVSNWLS?RITTQFNHIN
YQMYDSLSYQ?ADAIKAKIDL?EYKKYSGSDK?ENIKSQVENL?KNSLDVKISE?AMNNINKFIR
ECSVTYLFKN?MLPKVIDELN?KFDLRTKTEL?INLIDSHNII?LVGEVDRLKA?KVNESFENTM
PFNIFSYTNN?SLLKDIINEY?FNSINDSKIL?SLQNKKNALV?DTSGYNAEVR?VGDNVQLNTI
YTNDFKLSSS?GDKIIVNLNN?NILYSAIYEN?SSVSFWIKIS?KDLTNSHNEY?TIINSIEQNS
GWKLCIRNGN?IEWILQDVNR?KYKSLIFDYS?ESLSHTGYTN?KWFFVTITNN?IMGYMKLYIN
GELKQSQKIE?DLDEVKLDKT?IVFGIDENID?ENQMLWIRDF?NIFSKELSNE?DINIVYEGQI
LRNVIKDYWG?NPLKFDTEYY?IINDNYIDRY?IAPESNVLVL?VQYPDRSKLY?TGNPITIKSV
SDKNPYSRIL?NGDNIILHML?YNSRKYMIIR?DTDTIYATQG?GECSQNCVYA?LKLQSNLGNY
GIGIFSIKNI?VSKNKYCSQI?FSSFRENTML?LADIYKPWRF?SFKNAYTPVA?VTNYETKLLS
TSSFWKFISR?DPGWVE(SEQ?ID?NO：142)
5.E type botulic neurotoxin (BoNT/E) (EC 3.4.24.69) (Bontoxilysin-E)
Q00496；BXE_CLOBO
MPKINSFNYN?DPVNDRTILY?IKPGGCQEFY?KSFNIMKNIW?IIPERNVIGT?TPQDFHPPTS
LKNGDSSYYD?PNYLQSDEEK?DRFLKIVTKI?FNRINNNLSG?GILLEELSKA?NPYLGNDNTP
DNQFHIGDAS?AVEIKFSNGS?QDILLPNVII?MGAEPDLFET?NSSNISLRNN?YMPSNHRFGS
IAIVTFSPEY?SFRFNDNCMN?EFIQDPALTL?MHELIHSLHG?LYGAKGITTK?YTITQKQNPL
ITNIRGTNIE?EFLTFGGTDL?NIITSAQSND?IYTNLLADYK?KIASKLSKVQ?VSNPLLNPYK
DVFEAKYGLD?KDASGIYSVN?INKFNDIFKK?LYSFTEFDLR?TKFQVKCRQT?YIGQYKYFKL
SNLLNDSIYN?ISEGYNINNL?KVNFRGQNAN?LNPRIITPIT?GRGLVKKIIR?FCKNIVSVKG
IRKSICIEIN?NGELFFVASE?NSYNDDNINT?PKEIDDTVTS?NNNYENDLDQ?VILNFNSESA
PGLSDEKLNL?TIQNDAYIPK?YDSNGTSDIE?QHDVNELNVF?FYLDAQKVPE?GENNVNLTSS
IDTALLEQPK?IYTFFSSEFI?NNVNKPVQAA?LFVSWIQQVL?VDFTTEANQK?STVDKIADIS
IVVPYIGLAL?NIGNEAQKGN?FKDALELLGA?GILLEFEPEL?LIPTILVFTI?KSFLGSSDNK
NKVIKAINNA?LKERDEKWKE?VYSFIVSNWM?TKINTQFNKR?KEQMYQALQN?QVNAIKTIIE
SKYNSYTLEE?KNELTNKYDI?KQIENELNQK?VSIAMNNIDR?FLTESSISYL?MKIINEVKIN
KLREYDENVK?TYLLNYIIQH?GSILGESQQE?LNSMVTDTLN?NSIPFKLSSY?TDDKILISYF
NKFFKRIKSS?SVLNMRYKND?KYVDTSGYDS?NININGDVYK?YPTNKNQFGI?YNDKLSEVNI
SQNDYIIYDN?KYKNFSISFW?VRIPNYDNKI?VNVNNEYTII?NCMRDNNSGW?KVSLNHNEII
WTFEDNRGIN?QKLAFNYGNA?NGISDYINKW?IFVTITNDRL?GDSKLYINGN?LIDQKSILNL
GNIHVSDNIL?FKIVNCSYTR?YIGIRYFNIF?DKELDETEIQ?TLYSNEPNTN?ILKDFWGNYL
LYDKEYYLLN?VLKPNNFIDR?RKDSTLSINN?IRSTILLANR?LYSGIKVKIQ?RVNNSSTNDN
LVRKNDQVYI?NFVASKTHLF?PLYADTATTN?KEKTIKISSS?GNRFNQVVVM?NSVGNCTMNF
KNNNGNNIGL?LGFKADTVVA?STWYYTHMRD?HTNSNGCFWN?FISEEHGWQE?K(SEQ?ID?NO：143)
6.F type botulic neurotoxin (BoNT/F) (EC 3.4.24.69) (Bontoxilysin-F)
P30996；BXF_CLOBO
MPVAINSFNY?NDPVNDDTIL?YMQIPYEEKS?KKYYKAFEIM?RNVWIIPERN?TIGTNPSDFD
PPASLKNGSS?AYYDPNYLTT?DAEKDRYLKT?TIKLFKRINS?NPAGKVLLQE?ISYAKPYLGN
DHTPIDEFSP?VTRTTSVNIK?LSTNVESSML?LNLLVLGAGP?DIFESCCYPV?RKLIDPDVVY
DPSNYGFGSI?NIVTFSPEYE?YTFNDISGGH?NSSTESFIAD?PAISLAHELI?HALHGLYGAR
GVTYEETIEV?KQAPLMIAEK?PIRLEEFLTF?GGQDLNIITS?AMKEKIYNNL?LANYEKIATR
LSEVNSAPPE?YDINEYKDYF?QWKYGLDKNA?DGSYTVNENK?FNEIYKKLYS?FTESDLANKF
KVKCRNTYFI?KYEFLKVPNL?LDDDIYTVSE?GFNIGNLAVN?NRGQSIKLNP?KIIDSIPDKG
LVEKIVKFCK?SVIPRKGTKA?PPRLCIRVNN?SELFFVASES?SYNENDINTP?KEIDDTTNLN
NNYRNNLDEV?ILDYNSQTIP?QISNRTLNTL?VQDNSYVPRY?DSNGTSEIEE?YDVVDFNVFF
YLHAQKVPEG?ETNISLTSSI?DTALLEESKD?IFFSSEFIDT?INKPVNAALF?IDWISKVIRD
FTTEATQKST?VDKIADISLI?VPYVGLALNI?IIEAEKGNFE?EAFELLGVGI?LLEFVPELTI
PVILVFTIKS?YIDSYENKNK?AIKAINNSLI?EREAKWKEIY?SWIVSNWLTR?INTQFNKRKE
QMYQALQNQV?DAIKTAIEYK?YNNYTSDEKN?RLESEYNINN?IEEELNKKVS?LAMKNIERFM
TESSISYLMK?LINEAKVGKL?KKYDNHVKSD?LLNYILDHRS?ILGEQTNELS?DLVTSTLNSS
IPFELSSYTN?DKILIIYFNR?LYKKIKDSSI?LDMRYENNKF?IDISGYGSNI?SINGNVYIYS
TNRNQFGIYN?SRLSEVNIAQ?NNDIIYNSRY?QNFSISFWVR?IPKHYKPMNH?NREYTIINCM
GNNNSGWKIS?LRTVRDCEII?WTLQDTSGNK?ENLIFRYEEL?NRISNYINKW?IFVTITNNRL
GNSRIYINGN?LIVEKSISNL?GDIHVSDNIL?FKIVGCDDET?YVGIRYFKVF?NTELDKTEIE
TLYSNEPDPS?ILKNYWGNYL?LYNKKYYLFN?LLRKDKYITL?NSGILNINQQ?RGVTEGSVFL
NYKLYEGVEV?IIRKNGPIDI?SNTDNFVRKN?DLAYINVVDR?GVEYRLYADT?KSEKEKIIRT
SNLNDSLGQI?IVMDSIGNNC?TMNFQNNNGS?NIGLLGFHSN?NLVASSWYYN?NIRRNTSSNG
CFWSSISKEN?GWKE(SEQ?ID?NO：144)
7.G type botulic neurotoxin (BoNT/G) (EC 3.4.24.69) (Bontoxilysin-G)
Q60393；BXG_CLOBO
MPVNIKXFNY?NDPINNDDII?MMEPFNDPGP?GTYYKAFRII?DRIWIVPERF?TYGFQPDQFN
ASTGVFSKDV?YEYYDPTYLK?TDAEKDKFLK?TMIKLFNRIN?SKPSGQRLLD?MIVDAIPYLG
NASTPPDKFA?ANVANVSINK?KIIQPGAEDQ?IKGLMTNLII?FGPGPVLSDN?FTDSMIMNGH
SPISEGFGAR?MMIRFCPSCL?NVFNNVQENK?DTSIFSRRAY?FADPALTLMH?ELIHVLHGLY
GIKISNLPIT?PNTKEFFMQH?SDPVQAEELY?TFGGHDPSVI?SPSTDMNIYN?KALQNFQDIA
NRLNIVSSAQ?GSGIDISLYK?QIYKNKYDFV?EDPNGKYSVD?KDKFDKLYKA?LMFGFTETNL
AGEYGIKTRY?SYFSEYLPPI?KTEKLLDNTI?YTQNEGFNIA?SKNLKTEFNG?QNKAVNKEAY
EEISLEHLVI?YRIAMCKPVM?YKNTGKSEQC?IIVNNEDLFF?IANKDSFSKD?LAKAETIAYN
TQNNTIENNF?SIDQLILDND?LSSGIDLPNE?NTEPFTNFDD?IDIPVYIKQS?ALKKIFVDGD
SLFEYLHAQT?FPSNIENLQL?TNSLNDALRN?NNKVYTFFST?NLVEKANTVV?GASLFVNWVK
GVIDDFTSES?TQKSTIDKVS?DVSIIIPYIG?PALNVGNETA?KENFKNAFEI?GGAAILMEFI
PELIVPIVGF?FTLESYVGNK?GHIIMTISNA?LKKRDQKWTD?MYGLIVSQWL?STVNTQFYTI
KERMYNALNN?QSQAIEKIIE?DQYNRYSEED?KMNINIDFND?IDFKLNQSIN?LAINNIDDFI
NQCSISYLMN?RMIPLAVKKL?KDFDDNLKRD?LLEYIDTNEL?YLLDEVNILK?SKVNRHLKDS
IPFDLSLYTK?DTILIQVFNN?YISNISSNAI?LSLSYRGGRL?IDSSGYGATM?NVGSDVIFND
IGNGQFKLNN?SENSNITAHQ?SKFVVYDSMF?DNFSINFWVR?TPKYNNNDIQ?TYLQNEYTII
SCIKNDSGWK?VSIKGNRIIW?TLIDVNAKSK?SIFFEYSIKD?NISDYINKWF?SITITNDRLG
NANIYINGSL?KKSEKILNLD?RINSSNDIDF?KLINCTDTTK?FVWIKDFNIF?GRELNATEVS
SLYWIQSSTN?TLKDFWGNPL?RYDTQYYLFN?QGMQNIYIKY?FSKASMGETA?PRTNFNNAAI
NYQNLYLGLR?FIIKKASNSR?NINNDNIVRE?GDYIYLNIDN?ISDESYRVYV?LVNSKEIQTQ
LFLAPINDDP?TFYDVLQIKK?YYEKTTYNCQ?ILCEKDTKTF?GLFGIGKFVK?DYGYVWDTYD
NYFCISQWYL?RRISENINKL?RLGCNWQFIP?VDEGWTE(SEQ?ID?NO：145)
8. tetanus toxin (EC 3.4.24.68) (Tentoxylysin)
P04958；TETX_CLOTE
MPITINNFRY?SDPVNNDTII?MMEPPYCKGL?DIYYKAFKIT?DRIWIVPERY?EFGTKPEDFN
PPSSLIEGAS?EYYDPNYLRT?DSDKDRFLQT?MVKLFNRIKN?NVAGEALLDK?IINAIPYLGN
SYSLLDKFDT?NSNSVSFNLL?EQDPSGATTK?SAMLTNLIIF?GPGPVLNKNE?VRGIVLRVDN
KNYFPCRDGF?GSIMQMAFCP?EYVPTFDNVI?ENITSLTIGK?SKYFQDPALL?LMHELIHVLH
GLYGMQVSSH?EIIPSKQEIY?MQHTYPISAE?ELFTFGGQDA?NLISIDIKND?LYEKTLNDYK
AIANKLSQVT?SCNDPNIDID?SYKQIYQQKY?QFDKDSNGQY?IVNEDKFQIL?YNSIMYGFTE
IELGKKFNIK?TRLSYFSMNH?DPVKIPNLLD?DTIYNDTEGF?NIESKDLKSE?YKGQNMRVNT
NAFRNVDGSG?LVSKLIGLCK?KIIPPTNIRE?NLYNRTASLT?DLGGELCIKI?KNEDLTFIAE
KNSFSEEPFQ?DEIVSYNTKN?KPLNFNYSLD?KIIVDYNLQS?KITLPNDRTT?PVTKGIPYAP
EYKSNAASTI?EIHNIDDNTI?YQYLYAQKSP?TTLQRITMTN?SVDDALINST?KIYSYFPSVI
SKVNQGAQGI?LFLQWVRDII?DDFTNESSQK?TTIDKISDVS?TIVPYIGPAL?NIVKQGYEGN
FIGALETTGV?VLLLEYIPEI?TLPVIAALSI?AESSTQKEKI?IKTIDNFLEK?RYEKWIEVYK
LVKAKWLGTV?NTQFQKRSYQ?MYRSLEYQVD?AIKKIIDYEY?KIYSGPDKEQ?IADEINNLKN
KLEEKANKAM?ININIFMRES?SRSFLVNQMI?NEAKKQLLEF?DTQSKNILMQ?YIKANSKFIG
ITELKKLESK?INKVFSTPIP?FSYSKNLDCW?VDNEEDIDVI?LKKSTILNLD?INNDIISDIS
GFNSSVITYP?DAQLVPGING?KAIHLVNNES?SEVIVHKAMD?IEYNDMFNNF?TVSFWLRVPK
VSASHLEQYG?TNEYSIISSM?KKHSLSIGSG?WSVSLKGNNL?IWTLKDSAGE?VRQITFRDLP
DKFNAYLANK?WVFITITNDR?LSSANLYING?VLMGSAEITG?LGAIREDNNI?TLKLDRCNNN
NQYVSIDKFR?IFCKALNPKE?IEKLYTSYLS?ITFLRDFWGN?PLRYDTEYYL?IPVASSSKDV
QLKNITDYMY?LTNAPSYTNG?KLNIYYRRLY?NGLKFIIKRY?TPNNEIDSFV?KSGDFIKLYV
SYNNNEHIVG?YPKDGNAFNN?LDRILRVGYN?APGIPLYKKM?EAVKLRDLKT?YSVQLKLYDD
KNASLGLVGT?HNGQIGNDPN?RDILIASNWY?FNHLKDKILG?CDWYFVPTDE?GWTND(SEQ?IDNO：146)
9. diphtheria toxin, diphtherotoxin (DT) (NAD (+)-diphthamide ADP ribosyltransferase) (EC 2.4.2.36)
P00588；DTX_CORBE
MLVRGYVVSR?KLFASILIGA?LLGIGAPPSA?HAGADDVVDS?SKSFVMENFS?SYHGTKPGYV
DSIQKGIQKP?KSGTQGNYDD?DWKGFYSTDN?KYDAAGYSVD?NENPLSGKAG?GVVKVTYPGL
TKVLALKVDN?AETIKKELGL?SLTEPLMEQV?GTEEFIKRFG?DGASRVVLSL?PFAEGSSSVE
YINNWEQAKA?LSVELEINFE?TRGKRGQDAM?YEYMAQACAG?NRVRRSVGSS?LSCINLDWDV
IRDKTKTKIE?SLKEHGPIKN?KMSESPNKTV?SEEKAKQYLE?EFHQTALEHP?ELSELKTVTG
TNPVFAGANY?AAWAVNVAQV?IDSETADNLE?KTTAALSILP?GIGSVMGIAD?GAVHHNTEEI
VAQSIALSSL?MVAQAIPLVG?ELVDIGFAAY?NFVESIINLF?QVVHNSYNRP?AYSPGHKTQP
FLHDGYAVSW?NTVEDSIIRT?GFQGESGHDI?KITAENTPLP?IAGVLLPTIP?GKLDVNKSKT
HISVNGRKIR?MRCRAIDGDV?TFCRPKSPVY?VGNGVHANLH?VAFHRSSSEK?IHSNEISSDS
IGVLGYQKTVDHTKVNSKLS?LFFEIKS(SEQ?ID?NO：147)
10. pseudomonas extracellular toxin
P11439；TOXA_PSEAE
MHLTPHWIPL?VASLGLLAGG?SFASAAEEAF?DLWNECAKAC?VLDLKDGVRS?SRMSVDPAIA
DTNGQGVLHY?SMVLEGGNDA?LKLAIDNALS?ITSDGLTIRL?EGGVEPNKPV?RYSYTRQARG
SWSLNWLVPI?GHEKPSNIKV?FIHELNAGNQ?LSHMSPIYTI?EMGDELLAKL?ARDATFFVRA
HESNEMQPTL?AISHAGVSVV?MAQAQPRREK?RWSEWASGKV?LCLLDPLDGV?YNYLAQQRCN
LDDTWEGKIY?RVLAGNPAKH?DLDIKPTVIS?HRLHFPEGGS?LAALTAHQAC?HLPLETFTRH
RQPRGWEQLE?QCGYPVQRLV?ALYLAARLSW?NQVDQVIRNA?LASPGSGGDL?GEAIREQPEQ
ARLALTLAAA?ESERFVRQGT?GNDEAGAASA?DVVSLTCPVA?AGECAGPADS?GDALLERNYP
TGAEFLGDGG?DISFSTRGTQ?NWTVERLLQA?HRQLEERGYV?FVGYHGTFLE?AAQSIVFGGV
RARSQDLDAI?WRGFYIAGDP?ALAYGYAQDQ?EPDARGRIRN?GALLRVYVPR?SSLPGFYRTG
LTLAAPEAAG?EVERLIGHPL?PLRLDAITGP?EEEGGRLETI?LGWPLAERTV?VIPSAIPTDP
RNVGGDLDPS?SIPDKEQAIS?ALPDYASQPG?KPPREDLK(SEQ?ID?NO：148)
Peptide is synthetic
At least have four kinds of modes that obtain peptide: (1) is from living things system (for example, tissue, serum, urine etc.) purification; (Donini P etc., Acta Endocrinol (Copenh) .1966; 52 (2): 169-85 and Donini P etc., Acta Endocrinol (Copenh) .1966; 52 (2): 186-98)); (2) purified peptide fragment after protein digestibility; (Schulz-Knappe P etc., Eur J Med Res.1996; 1 (5): 223-36 and Kilara A. and Panyam D.Crit Rev Food Sci Nutr.2003; 43 (6): 607-33)); (3) genetic engineering well known in the art and recombinant technique (Martial JA etc., Science.1979; 205 (4406): 602-7)); And (4) direct chemical synthetic (Peptide Synthesis and Applications, 1984, compile (Methods in Molecular Biology by John Howl; The 298th volume), Humana Press, Totowa; NJ.Chemistry of Peptide Synthesis, 2005, N.Leo Benoiton; CRC publishing house, Boca Raton, FL).
Owing to lack the control to peptide sequence, two methods are infeasible usually before the institute.Because the concentration of peptide is lower in the biological sample, need be at a considerable amount of concentration steps before the purification, so first method also is problematic.Therefore, synthetic concerning direct chemical than short peptide usually is attractive selection, and recombinant technique is preferred concerning bigger peptide.
In view of the complexity of aminoacid and peptide, traditional organic chemistry synthetic method is concerning having more than normally infeasible the peptide of four or five amino acid residue.Problem is included in and has a plurality of reactive groups in the peptide, and said a plurality of reactive groups cause a plurality of sites of puting together on the peptide, is impure thereby cause peptide mixer with respect to target peptide, therefore after each step, need carry out purification.(reference: Lehninger Principles of Biochemistry, the 3rd edition, 2000, compile by David L.Nelson and Michael M.Cox, Worth Publishers, New York, NY).
The synthetic aspect of the direct chemical that appears at peptide of solid-phase peptide synthetic (Merrifield, 1962) provides important breakthrough, wherein in said solid-phase peptide is synthetic, in synthetic peptide, said peptide is fixed in an end of solid carrier.Now, the synthetic FMOC chemical action that relates to of most of solid-phase peptide.Briefly, chemosynthesis proceeds to aminoterminal (N end) from c-terminus (C end).Solid phase carrier is insoluble polymer or resin.Unwanted reaction takes place at the alpha-amido place of amino acid residue in the prevention of 9-fluorenyl-methyl ester (FMOC) group.In the repetition period, use standard set to be reflected at every next aminoacid structure peptide on the resin carrier.The alpha-amino C terminal amino acid that at first, will have by the FMOC radical protection is connected on the reactive group on the resin.Usually use gentle organic base, the protection base on the amino acid whose alpha-amido that is connected to resin is removed.Now, second aminoacid that has the resin preparation reception peptide of C terminal amino acid.Receive each aminoacid, use different chemical actions to locate to protect said aminoacid simultaneously at alpha-amido (FMOC) and carboxyl (being generally dicyclohexylcarbodiimide (DCC)).Activate second amino acid whose carboxyl through removing DCC, and make on said second amino acid whose carboxyl and the solid carrier first amino acid whose de-protected alpha-amido reaction with formation peptide bond (Peptide Synthesis and Applications, 1984; Compile (Methods in Molecular Biology, the 298th volume), Humana Press by John Howl; Totowa, NJ.Chemistry of Peptide Synthesis, 2005; N.Leo Benoiton; CRC Press, Boca Raton, FL).(reference: Lehninger Principles of Biochemistry, the 3rd edition, 2000, compile by David L.Nelson and Michael M.Cox, Worth Publishers, New York, NY).
In each consecutive steps in circulation, the protection chemical group is blocked unwanted reaction and according to following order: (i) alpha-amido on the nascent peptide is gone protection; (ii) activate the carboxyl on the next aminoacid; And the reaction that (iii) continues to form peptide bond, up to synthetic whole peptide sequence.When the synthetic completion of peptide, the binding between cracking resin and the peptide is to obtain final peptide.State-of-the-art solid-phase peptide synthetic technology is automatically, and can obtain now and at well known some kinds of commercialization instruments.(Peptide Synthesis and Applications, 1984, compile (Methods in Molecular Biology by John Howl; The 298th volume), Humana Press, Totowa; NJ.Chemistry of Peptide Synthesis, 2005, N.Leo Benoiton; CRC Press, Boca Raton, FL; Lehninger Principles of Biochemistry, the 3rd edition, 2000, compile by David L.Nelson and Michael M.Cox, Worth Publishers, New York, NY).
Because solid phase synthesis is the progressively processing that is used for longer peptide, reduces gross production rate and thereby increase the critical limitation of cost so it has.For example, under the situation of the progressively productive rate 96%, the gross production rate of 21mer, 51mer and 100mer is respectively 44%, 13% and 1.7%.Similarly, under the situation of the progressively productive rate 99.8%, the gross production rate of 21mer, 51mer and 100mer is respectively 96%, 90% and 82%.Therefore; Concerning longer peptide; Making the A sequence gene through engineering approaches in the expression cassette and expressing sequence in suitable expression system (for example, microbial expression system, for example escherichia coli or yeast) or the mammalian expression systems (cell culture) is that cost is effectively and time efficient more.Yet, concerning less peptide, compare with solid-phase peptide is synthetic, make the cost of sequence gene through engineering approaches and expression and purified peptide be generally the effective and time efficient of cost.
Use said method or known in the art other is synthetic, expression or purification process synthesizes, expression or purification can be used for peptide of the present invention.
The formation of dsRNA-peptide conjugate
At least one peptide and dsRNA on first chain or on second chain or both and put together at 3 ' end or at 5 ' end or both or inside.Can peptide of the present invention and dsRNA of the present invention be puted together through any amino acid residue in the peptide; For example; Put together through the carboxyl of C terminal amino acid and the C terminal amino acid of C end; Or the N terminal amino acid of alpha-amido through the N terminal amino acid and N end is puted together or put together with particular functional group's (for example, Cys last-amino of SH base or Lys) on the amino acid residue.
Use peptide or the proteinic any chemical action of puting together of being used for known in the art, dsRNA and peptide of the present invention are puted together (reference: Bioconjugate Techniques, 1996, Greg T.Hermanson, Academic Press, San Diego, CA.; Chemistry of Protein Conjugation and Cross-linking, 1991, Shan S.Wong, CRC Press, Boca Raton, FL).
In one embodiment, with (CH 2) 6-NH 35 ' end of synthetic first chain of connexon or second chain, and use the maleimide chemical action will 5 of said first chain or second chain ' end and the Cys of peptide-the SH group puts together, to form stable conjugate.
In another embodiment, with (CH 2) 63 ' end of synthetic first chain of-SH connexon or second chain, and through disulfide exchange will 3 of said first chain or second chain ' end and Cys-SH group or peptide put together, with the conjugate of formation cleavable.
After puting together, through method purification dsRNA-peptide conjugate (Oehlke J etc., Eur J Biochem.2002 well known in the art; 269 (16): 4025-32, Hamma T and Miller PS.Bioconjug Chem.2003; 14 (2): 320-30, Zatsepin TS etc., Bioconjug Chem.2005; 16 (3): 471-89, Ferenc G etc., Nucleosides Nucleotides Nucleic Acids.2005; 24 (5-7): 1059-61), and the analytical method of the standard of use characterizes the homogeneity and the purity of dsRNA-peptide conjugate.
Confirm that dsRNA sends the function of peptide conjugate
Analyze dsRNA-peptide conjugate of the present invention to confirm that dsRNA is delivered to suitable target and the cracked ability of mediate rna i (like hereinafter acceptance of the bid inscribe one's name the chapters and sections that are called " RNAi In Vitro Assay to Assess DsiRNA Activity " described in).Also analyze dsRNA peptide conjugate of the present invention to confirm with the ability of delivery of peptides to suitable target.
In one embodiment, dsRNA-peptide or independent peptide are connected to cell surface, dsRNA peptide or independent peptide and cell surface are interacted.Through direct permeates cell membranes, through endocytic pathway, through above two kinds of methods or through other method known in the art, absorb dsRNA-peptide conjugate or independent peptide by cell.
Can confirm the function of dsRNA-peptide conjugate of the present invention according to following method through quantitative dsRNA oligonucleotide.
Be used for quantitatively comprising the solid phase extractions that is used for separate analytes and matrix, carry out reversed phase ion pairing Ultra Performance Liquid Chromatography (UPLC) separation and detection through electrospray ionization tandem mass spectrum analytic process (ESI-MS/MS) subsequently from the technology of the DsiRNA oligonucleotide of blood plasma or tissue sample.Analysis and test device comprises the Waters Acquity UPLC chromatograph with photodiode array detector, and this Waters Acquity UPLC chromatograph and three grades of QMSs of Waters Quattro Premiere are connected in series.
Use Phenomenex ' s Clarity extraction medium and scheme to accomplish solid phase extractions." load/dissolving " buffer added to contain oligonucleotide plasma sample to remove any bonded protein.Preferably, oligonucleotide is adsorbed onto on the solid-phase media.Subsequently, will suppress to separate and Ionized impurity and salt to remove with a series of buffer washing oligonucleotide.Finally, with oligonucleotide from medium eluting, concentrate and resuspending in buffer, this receives the check of downstream analysis.
Use the mobile phase and the C of hexafluoroisopropanol (HFIP) and triethylamine (TEA) 18Static phase is accomplished chromatography.Use electrospray ionization to accomplish Mass Spectrometer Method, the MS (MS/MS) that connects subsequently analyzes.Exploitation LC/MS system changes with the characteristic of confirming the specific oligonucleotides molecule.Under the concentration that changes, compare quantitative (Lin etc., J Pharm Biomed Anal.2007 June 28: 44 (2): 330-341) of the DsiRNA content accomplished in the sample through standard curve with identical DsiRNA in the MS response of sample and the specimen.The DsiRNA oligonucleotide mass concentration that final data is expressed as the per unit volume sample (for example, ng/mL).
The modification of DsiRNA
With dsRNA and dsRNA-peptide conjugate in-vitro transfection in the cell culture object model with the relatively picked-up of setting up dsRNA and dsRNA-peptide conjugate or send.Use suitable cell culture object model, and the terminal point measurement includes but not limited to one or more in following: (i) use the mRNA of qPCR quantitative; (ii) use the quantification of protein of Western blotting; The (iii) labeled cell internalization of dsRNA and dsRNA-peptide conjugate.To the delivery rate of the amount of sending dsRNA, dsRNA and the stability of sending dsRNA, for example use above-mentioned terminal point to measure, assess the relatively picked-up of dsRNA and dsRNA-peptide conjugate or send.
In an example, in being used for, carry out transfection with 24 orifice plates of dsRNA or dsRNA-peptide conjugate transfection to HeLa cell or 48 orifice plates.Before using, dsRNA and dsRNA-peptide conjugate are diluted in the cell culture medium, and at room temperature hatch about 30min.Concerning dose response experiments, the ultimate density of applied dsRNA and dsRNA-peptide conjugate changes in 0 to 50nM scope.Concerning time-process experiment, in order to confirm as the employed speed of dsRNA of sending defined herein that research is according to the optium concentration of the determined dsRNA-peptide conjugate of dose response experiments in various incubation times (for example, 30 minutes to 7 days).
Also through with fluorescence labels difference labelling peptide and dsRNA with carry out fluorescence Position Research altogether, the function of coming test peptides, dsRNA and dsRNA-peptide conjugate.With green fluorescence dye marker peptide, and with red fluorescence dyestuff labelling dsRNA.Use this method and make comparisons and confirm that peptide makes the ability of independent peptide and dsRNA-peptide conjugate internalization with the location of free (that is, unconjugated) dsRNA.Below with reference to document description how to carry out fluorescence location and cell transportation research-Moschos etc., Bioconjug Chem.2007; 18 (5): 1450-1459; Moschos etc., Biochemical Society Transactions 2007; 35 (4): 807-810; Lord-Fontaine etc., J.Neurotrauma 2008; 25:1309-1322; Winton etc., J.Biol Chem.2002; 36 (6): 32820-32829; Lu, Langer and Chen.Mol Pharm.2009 March 30 [before publication, being published on the network]; McNaughton etc., Proc Natl Acad Sci U S A.2009; 106 (15): 6111-6116.
The modification of dsRNA
A principal element that suppresses double-stranded RNA (" dsRNA ") effect is that dsRNA (for example, double-stranded RNA, siRNA and DsiRNA) passes through nuclease degradation.3 ' exonuclease is the main nuclease that is present in the serum, and the modification of 3 ' end of antisence strand dna oligonucleotide is most important to preventing to degrade.(Eder etc., 1991, Antisense Res Dev, 1:141-151).The nuclease of ribonuclease T family has been identified as so-called ERI-1, and this ERI-1 has 3 of the adjusting of participating in siRNA and degraded ' to 5 ' exonuclease activity (Kennedy etc., 2004, Nature 427:645-649; Hong etc., 2005, Biochem J, 390:675-679).This gene also is called Thex1 (NM_02067) or is called THEX1 (NM_153332) at philtrum in mice, and said gene is participated in the degraded of histone mRNA; Said gene also mediates among the siRNA 3 ' outstanding degraded but the duplex RNA of not degrading (Yang etc., 2006, J Biol Chem, 281:30447-30454).Therefore, expect that it is rational that 3 of dsRNA (comprising DsiRNA of the present invention) ' end stabilisation will improve stability.
XRN1 (NM_019001) for be present in handle in the corpusculum (P-body) 5 ' to 3 ' exonuclease and with the degraded (Rehwinkel etc. that relate to the mRNA of miRNA targeting; 2005; RNA 11:1640-1647), and said XRN1 (NM_019001) can also be responsible for accomplishing the degraded that causes by inner cracking by the siRNA guiding.XRN2 (NM_012255) is for participating in 5 ' to 3 ' exonuclease of nRNA processing.Though current degraded or the processing of not participating in siRNA and miRNA, but both are all known nucleic acid enzyme and this also emphasis consideration of degradable RNA.
Ribonuclease A is the main endonuclease activity in the mammal of degradation of rna.It has specificity to ssRNA and in 3 ' end place cracking of pyrimidine bases.Can detect the SiRNA catabolite (Turner etc., 2007, Mol Biosyst 3:43-50) that conforms to the ribonuclease A cracking through mass spectrography after in serum, hatching.3 ' outstanding the susceptibility that strengthens siRNA to the ribonucleic acid enzymatic degradation.Exhausted from the reduction of the ribonuclease A of serum the degraded of siRNA; This degraded shows some sequence preferences really and on endways, has unfavorable (Haupenthal etc., 2006Biochem Pharmacol 71:702-710) the sequence of gathering the A/U sequence.This has shown following probability: the transition strand kind that can supply the ribonuclease A degraded can " be breathed " and provided to the low stable region of duplex.The ribonuclease A inhibitor can add in the serum and improve life-span and the effectiveness (Haupenthal etc., 2007, Int J.Cancer 121:206-210) of siRNA.
In 21mer, thiophosphate or borine phosphate ester are modified and are directly made between nucleoside phosphate bond stable.The RNA high resistance nuclease that the borine phosphate ester is modified is effective and nontoxic relatively as reticent agent.Can not use the standard learning synthetic method to prepare the RNA that the borine phosphate ester is modified, but prepare (Hall etc., 2004, Nucleic Acids Res 32:5991-6000 through in vitro transcription (IVT); Hall etc., 2006, Nucleic Acids Res 34:2773-2781).Thiophosphate (PS) is modified can be easy to be placed on any desired position in the RNA duplex, and can use the preparation of standard learning synthetic method.PS modifies the toxicity that demonstrates dependent dose; So most researchers recommends to incorporate into siRNA limitedly; It is most important (Harborth etc., 2003, Antisense Nucleic Acid Drug Dev 13:83-105 that approval prevents to receive the nuclease influence at 3 ' end; Chiu and Rana, 2003, Mol Cell 10:549-561; Braasch etc., 2003, Biochemistry 42:7967-7975; Amarzguiou etc., 2003, Nucleic Acids Research 31:589-595).Widely PS modify can with effective RNAi activity compatible; Yet, use sugar-modified (such as, 2 '-the O-methyl RNA) maybe more excellent (Choung etc., 2006, Biochem Biophys Res Commun342:919-927).
Various replacements can be positioned at 2 ' position of ribose, and this improves duplex stability (Tm) usually and can greatly improve the nuclease resistance.2 '-the O-methyl RNA is the natural modifications that is found in mammal nuclear candy body RNA and the transfer RNA.Among the siRNA 2 '-O-methyl is modified to known, but the exact position of modified base is very important to maintenance effectiveness in the duplex, and 2 '-the O-methyl RNA replaces RNA fully will make the siRNA inactivation.For example, adopt alternately 2 '-effectiveness of the pattern of O-methyl base can be with suitable without the effectiveness of modifying RNA, and said pattern very stable (Choung etc., 2006, Biochem Biophys Res Commun 342:919-927 in serum; Czauderna etc., 2003, Nucleic Acids Research 31:2705-2716).
2 '-fluorine (2 '-F) modification is also compatible with dsRNA (for example, siRNA and DsiRNA) function; The most generally 2 '-fluorine (2 '-F) modify and be positioned at pyrimidine site (this is because the cause of reagent cost and availability), and 2 '-fluorine (2 '-F) modify can with in 2 of purine position '-the O-methyl modifies and makes up; 2 '-the F purine can obtain and also can use.The a large amount of duplexs of modifying of this type can be effective triggering agent of external RNAi, (Allerson etc., 2005, J Med Chem 48:901-904; Prakash etc., 2005, J Med Chem 48:4247-4253; Kraynack and Baker, 2006, RNA 12:163-176), and when using in vivo, can improve persistent period (Morrissey etc., 2005, the Hepatology 41:1349-1356 of usefulness and prolongation effect; Morrissey etc., 2005, Nat Biotechnol 23:1002-1007).Allerson instructed contain substituting 2 '-F and 2 '-O-Me base efficiently, the stable flush end 19mer duplex of nuclease.In this design, with the identical pattern of pattern that Czauderna is adopted locate alternative 2 '-the O-Me residue, yet residue RNA residue convert 2 into '-the F modified base.The resistance of nuclease efficiently siRNA by Morrissey adopted adopts nuclease resistance siRNA efficiently in vivo.Except 2 '-O-Me RNA and 2 '-FRNA, this duplex comprises that also DNA, RNA, reversing do not have bonding between base residue and 3 ' end PS nucleoside.Have some benefit though extensively modify, the limited modification of duplex also can improve usefulness in vivo and more simply and more at low cost prepare.Soutschek etc. (2004, Nature 432:173-178) adopt duplex in vivo, and mainly be have two 2 '-O-Me RNA base and restricted 3 ' end PS nucleoside between the RNA of bonding.
Lock nucleic acid (LNA) for can be used for stabilizing dsrna (for example, siRNA and DsiRNA) different classes of 2 '-modification.The LNA that keep to render a service incorporate into pattern than 2 '-O-methyl or 2 '-the F base is more restricted, so preferred limited modification (Braasch etc., 2003, Biochemistry 42:7967-7975; Grunweller etc., 2003, Nucleic Acids Res 31:3185-3193; Elmen etc., 2005, Nucleic Acids Res33:439-447).Even under limited situation about incorporating into, use LNA to modify also can to improve dsRNA in vivo usefulness and can also change or improve the effect collection of illustrative plates (Mook etc., 2007, Mol Cancer Ther 6:833-843) that misses the target.
The synthesizing ribonucleotide that is introduced in cell or the live animal can be considered to " external " and trigger immunoreation.Immunostimulation constitutes the effect of missing the target of primary categories, and the said effect of missing the target can change experimental result significantly even cause cell death.Natural immune system comprises and the DNA of these reactions of mediation and the set of RNA specificity interaction receptor molecule; In the middle of them some are arranged in Cytoplasm and their central some are present in endosome (Marques and Williams; 2005, Nat Biotechnol 23:1399-1405; Schlee etc., 2006, Mol Ther 14:463-470).Make siRNA be exposed to Cytoplasm and endosome compartment through cation lipid or liposome delivery siRNA, trigger the risk maximum that 1 type disturbs (IFN) reaction in vitro and in vivo thereby make.(Morrissey etc., 2005, Nat Biotechnol 23:1002-1007; Sioud and Sorensen, 2003, Biochem Biophys Res Commun 312:1220-1225; Sioud, 2005, J Mol Biol 348:1079-1090; Ma etc., 2005, Biochem Biophys Res Commun 330:755-759).Lower (the Robbins etc. of intracellular transcribe rna immunity; 2006; Nat Biotechnol 24:566-571) and when being incorporated on the cell (even in body) through Mechanical Method; The synthetic RNA of immunogenicity can avoid immunostimulation (Heidel etc., 2004, Nat Biotechnol 22:1579-1582) when use is sent based on the lipid method.Yet the method for sending based on lipid is convenient, effective and widely used.Especially in body, use (wherein having all cells type and produce immunoreactive risk the highest), need some to prevent immunoreactive general strategy.Use the RNA of chemical modification can solve major part and even all these problems.
Though some sequence motifs obviously produces more immunity than other sequence motifs, seemingly the receptor of natural immune system generally can be distinguished with the existence of in former nRNA, comparing more normal some base modification of finding in mammalian rna or not exist.For example, pseudouridine, N6-methyl-A and 2 '-O-methyl base of modifying is considered to " self " and in synthetic RNA, comprises these residues to assist to avoid immune detection (Kariko etc., 2005, Immunity 23:165-175).When in mouse vein, using, as without the strong immunostimulating sequence of modifying RNA extensive 2 '-modify immunoreation capable of blocking (Morrissey etc., 2005, Nat Biotechnol 23:1002-1007).Yet extensively modifying is for avoiding immune detection institute unwanted, and in the strand of siRNA duplex few to two 2 '-replacement of O-methyl base can be enough to block 1 type IFN reaction in external and the body; Through the U base of modifying and G base is the most effective (Judge etc., 2006, Mol Ther13:494-505).As the benefit that increases, selectivity incorporates 2 into '-O-methyl base can reduce the amplitude (Jackson etc., 2006, RNA 12:1197-1205) of the effect of missing the target.Therefore, all dsRNA that in being used for body, use, should be with 2 '-use of O-methyl base regards the method for blocking immunity reaction as, and has the benefit that improves the nuclease stability and the increase of the probability that reduces the effect of missing the target.
Though cell death possibly be to be produced by immunostimulation, assessing cell viability is not to be enough to monitor the method for inducing the IFN reaction.IFN reaction can exist under the situation of cell death not having, and cell death possibly be to strike low the generation by target lacking under the situation that IFN triggers (for example, under the requisite situation of target gene pair cell vigor).Can in culture medium, directly measure relevant cell factor, and have the multiple commercial kit that carries out this type of conventional analysis.Though can measure many different immune effector molecules, enough realize the screening purpose usually at the testing level of transfection IFN-α, TNF-α and IL-6 after 4 hours and 24 hours.Cation lipid comprises that " the unique contrast of transfection reagent " is very important, because can trigger the immunoreation in some cell that does not have any nucleic acid load.To cell culture work, considered comprises the inductive contrast of IFN approach.No matter when in vivo administration of nucleic acid (in this case, the risk that triggers the IFN reaction is the highest) is tested immunostimulation and is absolutely necessary.
Can not stop DsiRNA reagent as the Dicer substrate as long as modify, then can modification be included within the DsiRNA reagent of the present invention.In one embodiment, carrying out one or more modifies to promote the Dicer processing of DsiRNA reagent.In second embodiment, carry out one or more modifications and can cause more effective RNAi to produce.In the 3rd embodiment, carry out one or more and modify to support stronger RNAi effect.In the 4th embodiment, carry out one or more modifications and can in waiting to be delivered to each DsiRNA reagent molecule of cell, produce bigger effectiveness.Can incorporate modification into 3 ' petiolarea, 5 ' petiolarea, 3 ' petiolarea and 5 ' petiolarea perhaps in each position in (in some instances) sequence.In view of above-mentioned restriction, can the modification of any number and any combination be incorporated in the DsiRNA reagent.Exist under the situation of multiple modification, they possibly be identical or different.Contain modification to base, sugar moieties, phosphoric acid skeleton and their combination.Can make arbitrary 5 ' end phosphorylation.
Instance to the desired modification of phosphoric acid skeleton comprises phosphate ester, comprises that methyl phosphorodithioate, thiophosphate ester and phosphotriester modify (such as, alkyl phosphotriester) etc.To the instance of the desired modification of sugar moieties comprise 2 '-the alkyl pyrimidine, such as 2 '-O-methyl, 2 '-fluorine, amino and deoxidation modification etc. (for example, referring to Amarzguioui etc., 2003, Nucleic Acids Research 31:589-595).To the instance of the desired modification of base comprise no base sugar, '-pyrimidine, 4-thiouracil, 5-bromouracil, 5-iodouracil and 5-(the amino pi-allyl of 3-)-uracil that the O-alkyl is modified etc.Can also incorporate lock nucleic acid or LNA into.Many other modifications are known, as long as and satisfy above-mentioned standard and also can use said other modification.Also at United States Patent (USP) the 5th, 684, No. 143, the 5th, 858, No. 988 and the 6th, 291, in No. 438 and the U.S.'s publication application 2004/0203145A1 number the instance of modifying is disclosed.At Herdewijn (2000; Antisense Nucleic Acid Drug Dev 10:297-310), Eckstein (2000; Antisense Nucleic Acid Drug Dev 10:117-21), Rusckowski etc. (2000; Antisense Nucleic Acid Drug Dev 10:333-345), Stein etc. (2001, Antisense Nucleic Acid Drug Dev 11:317-25); Vorobjev etc. disclose other modification in (2001, Antisense Nucleic Acid Drug Dev 11:77-85).
Can desired one or more modifications be incorporated in arbitrary chain.Make modification is arranged in the characteristic that DsiRNA reagent can greatly influence DsiRNA reagent, comprise give more multiple-effect power with stability, reduce toxicity, strengthen Dicer processing and make immunoreation reduce to minimum.In one embodiment, antisense strand or sense strand or antisense strand and sense strand have one or more 2 '-nucleotide that the O-methyl is modified.In another embodiment, antisense strand contain 2 '-nucleotide that the O-methyl is modified.In another embodiment, antisense strand contain and comprise 2 '-nucleotide that the O-methyl is modified 3 ' outstanding.That antisense strand can also comprise is other 2 '-nucleotide that the O-methyl is modified.
In certain embodiments of the invention, DsiRNA reagent has one or more and strengthens it by the characteristic through Dicer processing.According to these embodiments; DsiRNA reagent has sufficient length; So that its through Dicer processing to generate at least one in active siRNA and the following characteristic: (i) DsiRNA reagent is asymmetric, for example, on antisense strand, has 3 ' outstanding; (ii) DsiRNA reagent has 3 of modification ' end on sense strand, combines and be processed into the orientation of active siRNA with the Dicer of guiding dsRNA.According to this embodiment, the longest chain comprises 25 to 35 nucleotide among the dsRNA.In one embodiment, DsiRNA reagent is asymmetric, make sense strand comprise 25 to 28 nucleotide, and antisense strand comprises 25 to 30 nucleotide.Therefore, the dsRNA of gained has outstanding on 3 of antisense strand ' end.Outstanding is 1 to 4 nucleotide, for example 2 nucleotide.Sense strand also can have 5 ' phosphoric acid.
In other embodiments, through being positioned at the suitable modification agent of 3 of sense strand ' end, the sense strand of DsiRNA reagent is modified the processing for Dicer, that is, design DsiRNA reagent is with the orientation of guiding Dicer combination and processing.Suitable dressing agent comprise nucleotide (such as, deoxyribonucleotide, bi-deoxyribose nucleotide, acyclic nucleotide etc.) and steric hindrance molecule (such as, fluorescence molecule etc.).Acyclic nucleotide is present in 2 among the dNMP '-desoxyribofuranose base sugar usually with 2-hydroxyl-oxethyl methyl substituted.Other nucleotide modification agent can comprise 3 '-deoxyadenosine (cordycepin), 3 '-azido-3 '-AZT (AZT), 2 '; 3 '-didanosine (ddI), 2 ', 3 '-two deoxidations-3 '-sulfo-cytidine (3TC), 2 ', 3 '-two dehydrogenations-2 '; 3 '-videx (d4T) and 3 '-azido-3 '-AZT (AZT), 2 '; 3 '-two deoxidations-3 '-sulfo-cytidine (3TC) and 2 ', 3 '-two dehydrogenations-2 ', 3 '-monophosphic acid nucleotide of videx (d4T).In one embodiment, Deoxydization nucleotide is used as dressing agent.When using the nucleotide modification agent, replace the ribonucleotide on sense strand 3 ' end with 1 to 3 nucleotide modification agent or 2 nucleotide modification agent.When using steric hindrance to divide the period of the day from 11 p.m. to 1 a.m, the steric hindrance molecule is connected to the ribonucleotide at 3 ' end place of antisense strand.Therefore, chain length can not change along with incorporating into of dressing agent.In another embodiment, the orientation that two DNA bases in the replacement DsiRNA reagent are processed with the Dicer that guides antisense strand is contained in the present invention.In another embodiment of the invention; Get last two ribonucleotides of 3 of sense strand ' end with two terminal DNA bases; Thereby on 5 ' end of 3 of sense strand ' end and antisense strand, form the flush end of duplex, and two outstanding being positioned on 3 ' end of antisense strand of nucleotide RNA.This is at the asymmetrical array thing that has DNA on the flush end and on jag, have the RNA base.
Under biotic factor (such as, the condition of in the Cytoplasm of cell, finding) with the sense strand and the antisense strand annealing of DsiRNA reagent of the present invention.In addition; The sequence of DsiRNA reagent (especially; The sequence length of a sequence area antisense strand) is at least 19 nucleotide, wherein these nucleotide in 21 nucleotide districts of 3 of contiguous antisense strand ' end and with by target gene in the nucleotide sequence of the RNA that produced fully complementary.
DsiRNA reagent also can have following one or more other characteristics: (a) antisense strand has moving to right with respect to typical 21mer; (b) chain is maybe not can complementary fully; Promptly; Chain possibly contain simple mispairing pairing and (c) base modification (such as, lock nucleic acid) possibly be included in 5 of sense strand ' end.Use conventional art design " typical case " 21mer siRNA.In a kind of technology; A reagent in hoping reagent is with under the effective situation; Usually a plurality of sites of parallel testing or contain the storehouse that identical target is had specific several different siRNA duplexs; In the hope of finding that a kind of reagent is effective (Ji etc., 2003, FEBS Lett 552:247-252).Other technology is used design rule and probability (Schwarz etc., 2003, the Cell 115:199-208 of algorithm to have increased access to viable rna i effector molecule; Khvorova etc., 2003, Cell 115:209-216; Ui-Tei etc., 2004, Nucleic Acids Res 32:936-948; Reynolds etc., 2004, Nat Biotechnol 22:326-330; Krol etc., 2004, J Biol Chem 279:42230-42239; Yuan etc., 2004, Nucl Acids Res 32 (web page server distribution): W130-134; Boese etc., 2005, Methods Enzymol 392:73-96).Also developed the high flux of siRNA and selected (U.S.'s publication application 2005/0042641A1 number).Can also analyze potential target site (Heale etc., 2005, Nucleic Acids Res 33 (3): e30) through secondary structure prediction.Subsequently, this 21mer is used to design moves to right on 5 ' end of 21mer, to comprise 3 to 9 other nucleotide.The sequence of the nucleotide that these are other possibly have any sequence.In one embodiment, the ribonucleotide of interpolation is based on the sequence of target gene.Even in this embodiment, do not need complete complementation between target sequence and the antisense strand siRNA yet.
First oligonucleotide and second oligonucleotide of DsiRNA reagent of the present invention do not need complete complementation.They only need complementary basically with annealing under biotic factor be that Dicer provides substrate, thereby produce and the abundant complementary siRNA of target sequence.Lock nucleic acid or LNA are (Elmen etc., 2005, Nucleic Acids Res 33:439-447 well-known to those skilled in the art; Kurreck etc., 2002, Nucleic Acids Res30:1911-1918; Crinelli etc., 2002, Nucleic Acids Res 30:2435-2443; Braasch and Corey, 2001, Chem Biol 8:1-7; Bondensgaard etc., 2000, Chemistry 6:2687-2695; Wahlestedt etc., 2000, Proc Natl Acad Sci USA 97:5633-5638).In one embodiment, incorporate LNA at 5 ' end place of sense strand.In another embodiment, through design with on antisense strand, comprise 3 ' 5 ' end place of the sense strand of outstanding duplex incorporates LNA into.
In certain embodiments, DsiRNA reagent of the present invention has dissymmetrical structure, and wherein the length of sense strand is that the length of 25 base pairs and antisense strand is that 27 base pairs and having contain 3 of 2 bases ' outstanding.In other embodiments, this DsiRNA reagent with dissymmetrical structure also contains 2 Deoxydization nucleotides at 3 of sense strand ' end, substitutes two ribonucleotides.
Can connect some the DsiRNA reagent composition that contains two independent oligonucleotide through the 3rd structure.The 3rd structure will can not be blocked the activity of Dicer to DsiRNA reagent, and said the 3rd structure will can not disturb the orientation of the RNA that is transcribed by target gene to destroy.In one embodiment, the 3rd structure possibly be a cytotoxic compounds.That many suitable cytotoxic compounds are known in the art and can use.Perhaps, the 3rd structure can be the oligonucleotide that connects two oligonucleotide of DsiRNA reagent with certain mode, makes after two oligonucleotide annealing forming the DsiRNA compositions, to produce hairpin structure.Hairpin structure will can not be blocked the activity of Dicer to DsiRNA reagent, and will can not disturb the orientation of target RNA to destroy.
In certain embodiments, DsiRNA reagent of the present invention has the some characteristics of enhancing Dicer to its processing.According to these embodiments; DsiRNA reagent has sufficient length; So that its through Dicer processing to produce siRNA and following at least a characteristic: (i) DsiRNA reagent is asymmetric, for example, on sense strand, has 3 ' outstanding; (ii) DsiRNA reagent has 3 ' end through modifying on antisense strand, with the Dicer of guiding dsRNA combine and with the orientation that is processed into active siRNA.According to these embodiments, the longest chain comprises 25 to 30 nucleotide in the DsiRNA reagent.In one embodiment, sense strand comprises 25 to 30 nucleotide and antisense strand comprises 25 to 28 nucleotide.Therefore, gained dsRNA has outstanding on 3 of sense strand ' end.Outstanding is 1 to 4 nucleotide, such as 2 nucleotide.Antisense strand also can have 5 ' phosphoric acid.
In certain embodiments, the suitable modification agent at the 3 ' end place through being positioned at sense strand is modified the processing for Dicer with the sense strand of DsiRNA reagent, that is, design DsiRNA reagent combines with guiding Dicer and the orientation of processing.Suitable dressing agent comprise nucleotide (such as, deoxyribonucleotide, bi-deoxyribose nucleotide, acyclic nucleotide etc.) and steric hindrance molecule (such as, fluorescence molecule etc.).Acyclic nucleotide is present in 2 among the dNMP '-desoxyribofuranose base sugar usually with 2-hydroxyl-oxethyl methyl substituted.Other nucleotide modification agent can comprise: 3 '-deoxyadenosine (cordycepin), 3 '-azido-3 '-AZT (AZT), 2 '; 3 '-didanosine (ddI), 2 ', 3 '-two deoxidations-3 '-sulfo-cytidine (3TC), 2 ', 3 '-two dehydrogenations-2 '; 3 '-videx (d4T) and 3 '-azido-3 '-AZT (AZT), 2 '; 3 '-two deoxidations-3 '-sulfo-cytidine (3TC) and 2 ', 3 '-two dehydrogenations-2 ', 3 '-monophosphic acid nucleotide of videx (d4T).In one embodiment, Deoxydization nucleotide is used as dressing agent.When using the nucleotide modification agent, the ribonucleotide on 3 ' end of 1 to 3 nucleotide modification agent or 2 nucleotide modification agent replacement sense strands.When using steric hindrance to divide the period of the day from 11 p.m. to 1 a.m, the steric hindrance molecule is connected to the ribonucleotide of 3 of antisense strand ' end.Therefore, chain length can not change along with incorporating into of dressing agent.In another embodiment, the present invention expects and replaces two DNA bases among the dsRNA with the orientation of guiding Dicer processing.In another invention; Two terminal DNA bases (replacing two ribonucleotides) are positioned on 3 ' end of sense strand; Thereby on 3 ' end of 5 of antisense strand ' end and sense strand, form the flush end of duplex, and two outstanding being positioned on 3 ' end of antisense strand of nucleotide RNA.This is at the asymmetrical array thing that has DNA on the flush end and on jag, have the RNA base.
In some other embodiment, the suitable modification agent at the 3 ' end place through being positioned at antisense strand is modified the processing for Dicer with the antisense strand of DsiRNA reagent, that is, design DsiRNA reagent combines with guiding Dicer and the orientation of processing.Suitable dressing agent comprise nucleotide (such as, deoxyribonucleotide, bi-deoxyribose nucleotide, acyclic nucleotide etc.) and steric hindrance molecule (such as, fluorescence molecule etc.).Acyclic nucleotide replaces being present in usually 2 among the dNMP '-desoxyribofuranose base sugar with 2-'-hydroxyethoxy ylmethyl.Other nucleotide modification agent can comprise: 3 '-deoxyadenosine (cordycepin), 3 '-azido-3 '-AZT (AZT), 2 '; 3 '-didanosine (ddI), 2 ', 3 '-two deoxidations-3 '-sulfo-cytidine (3TC), 2 ', 3 '-two dehydrogenations-2 '; 3 '-videx (d4T) and 3 '-azido-3 '-AZT (AZT), 2 '; 3 '-two deoxidations-3 '-sulfo-cytidine (3TC) and 2 ', 3 '-two dehydrogenations-2 ', 3 '-monophosphic acid nucleotide of videx (d4T).In one embodiment, Deoxydization nucleotide is used as dressing agent.When using the nucleotide modification agent, the ribonucleotide on 3 ' end of 1 to 3 nucleotide modification agent or 2 nucleotide modification agent replacement antisense strands.When using steric hindrance to divide the period of the day from 11 p.m. to 1 a.m, the steric hindrance molecule is connected to the ribonucleotide of 3 of antisense strand ' end.Therefore, chain length can not change along with incorporating into of dressing agent.In another embodiment, the present invention expects and replaces two DNA bases among the dsRNA with the orientation of guiding Dicer processing.In another invention; Two terminal DNA bases (replacing two ribonucleotides) are positioned on 3 ' end of antisense strand; Thereby on 3 ' end of 5 of sense strand ' end and antisense strand, form the flush end of duplex, and two outstanding being positioned on 3 ' end of sense strand of nucleotide RNA.This also is at the asymmetrical array thing that has DNA on the flush end and on jag, have the RNA base.
Under biotic factor (such as, the condition of in the Cytoplasm of cell, finding), with sense strand and antisense strand annealing.In addition, the length of a sequence area in the sequence of dsRNA (especially, antisense strand) is at least 19 nucleotide, wherein 3 ' end of the contiguous antisense strand of these nucleotide and fully complementary with the nucleotide sequence of target RNA.
In addition, can make the optimization of DsiRNA agent structure, to guarantee to become the part of oligonucleotide the most effective inhibition of gene expression from the oligonucleotide fragment that the Dicer cracking produces.For example, in one embodiment of the invention, the 27-bp oligonucleotide of synthetic DsiRNA agent structure, wherein expection 21-bp to the 22-bp fragment with inhibition of gene expression is positioned on 3 ' end of antisense strand.The residue base that is positioned on 5 ' end of antisense strand will and will be dropped by the Dicer cracking.This possibly is homologous (that is, based on the sequence of target sequence) or non-homogeneous by cracked part, and add this by cracked part to extend nucleic acid chains.
US 2007/0265220 discloses 27mer DsiRNA and in serum, has demonstrated with similar 21mer siRNA compositions and compare the stability of raising, even there is not chemical modification.When combining with the interpolation of 5 ' phosphoric acid, the modification of DsiRNA reagent (such as, pattern as previously discussed with 2 '-the O-methyl RNA is included in the antisense strand) can improve the stability of DsiRNA reagent.5 ' phosphoric acid to be added in all chains of synthetic RNA duplex possibly be cheap physiology method and possibly be the nuclease stability of giving certain limited extent.The chemical modification pattern of DsiRNA reagent of the present invention is designed to strengthen the effect of this type of reagent.Therefore, this type of modification is designed to avoid reducing the effectiveness of DsiRNA reagent; Avoid interference the Dicer processing of DsiRNA reagent; Stability (reduction nuclease sensitivity) in the biofluid of raising DsiRNA reagent; Or block or avoid detecting by the inherent immunity system.Also this type of modification is designed to not be deleterious, and avoids the simple production that increases cost or influence these DsiRNA reagent of the present invention.
The RNAi external test is active with assessment DsiRNA
RNAi is reproduced in the DsiRNA construct that external test in the cell-free system can be used for assessing a targeting target RNA sequence.This mensuration comprises by Tuschl etc., 1999, and Genes and Development, 13,3191-3197 and Zamore etc., 2000, Cell, 101, the described system of 25-33, this system are suitable for using with the DsiRNA reagent to target RNA.The fruit bat extract that derives from the syncytium blastodisc is used at reconstruction in vitro RNAi active.Through using t7 rna polymerase to carry out in vitro transcription or produce target RNA through chemosynthesis from suitable target rna expression plasmid.Through under 90 ℃ buffer (such as, 100mM potassium acetate, 30mM HEPES-KOH, pH 7.4,2mM magnesium acetate) in hatched 1 minute; Under 37 ℃, in buffer, cultivated 1 hour subsequently; Made adopted DsiRNA chain and antisense DsiRNA chain annealing (for example, each 20uM), subsequently (for example at the dissolving buffer; 100mM potassium acetate, 30mM HEPES-KOH, pH 7.4,2mM magnesium acetate) in dilution is said that adopted DsiRNA chain and antisense DsiRNA chain arranged.Can monitor annealing through carrying out gel electrophoresis on the agarose gel in tbe buffer liquid and using ethidium bromide staining.Be used in the zero embryo's (this embryo is through floss removing film and dissolving) who collects on the fermented-molasses agar, prepare the fruit bat lysate to two hours ages from Oregon R fly.Make lysate centrifugal, and supernatant is separated.Mensuration comprises reactant mixture, and this reactant mixture contains 50% lysate [volume], RNA (10 to 50pM ultimate density) and contains 10% [volume] the dissolving buffer of DsiRNA (10nM ultimate density).Reactant mixture also contains every seed amino acid of 10mM creatine phosphate, 10ug/ml creatine phosphokinase, 100um GTP, 100uM UTP, 100uM CTP, 500uMATP, 5mM DTT, 0.1U/ μ L RNAin (Promega) and 100uM.The ultimate density of potassium acetate is adjusted to 100mM.Before adding RNA, make up each reactant in advance and under 25 ℃, hatched in advance 10 minutes, under 25 ℃, hatched again subsequently other 60 minutes on ice.Use 1.25 of 4 volumes * passive lysis buffer (Promega) that reaction is stopped.Analyze or other method as known in the art is measured target RNA cracking by RT-PCR, and with reaction in do not comprise that the control reaction of DsiRNA compares.
Perhaps, through exist [α- 32P] carry out in vitro transcription under the situation of CTP, prepare the target RNA of the inner marker that is used to measure, said target RNA is separated through the rotation chromatography through the G50Sephadex post, and under without situation about being further purified with said target RNA as target RNA.Randomly, use the T4 polynucleotide kinase at target RNA 5 ' end labelling 32P.Measure as stated, and make the target RNA and the specific RNA pyrolysis product that produce by RNAi visible on the autoradiograph of gel.Cracking percentage ratio is quantitatively confirmed by expression complete contrast RNA or from the PHOSPHOR (autoradiography) of the band of the RNA of control reaction (not containing the DsiRNA and the pyrolysis product that are produced by measuring).
In one embodiment; This mensuration is used to DsiRNA mediate rna i cracking and confirms the target site in the target RNA target; For example come the assay determination reaction through electrophoresis by labeled target rna; Or through the RNA trace, and through the cracking of other method well known in the art to the RNAi mediation of several DsiRNA construct screening targets RNA target.
The structure of DsiRNA-peptide reagent
In certain embodiments, dsRNA reagent of the present invention can have following any structure:
In this type of embodiment, dsRNA comprises:
5′-XXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5’
Wherein, " X "=RNA, the outstanding domain that " Y " is made up of 1 to 4 RNA monomer, said RNA monomer randomly be 2 '-O-methyl RNA monomer, " D "=DNA and " P "=peptide.Cochain is a sense strand, and chain is an antisense strand down.
In another this type of embodiment, dsRNA comprises:
5′-PXXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5’
Wherein, " X "=RNA, the outstanding domain that " Y " is made up of 1 to 4 RNA monomer, said RNA monomer randomly be 2 '-O-methyl RNA monomer, " D "=DNA and " P "=peptide.Cochain is a sense strand, and chain is an antisense strand down.
In another this type of embodiment, dsRNA comprises:
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-YXXXXXXXXXXXXXXXXXXXXXXXXXP-5’
Wherein, " X "=RNA, the outstanding domain that " Y " is made up of 1 to 4 RNA monomer, said RNA monomer randomly be 2 '-O-methyl RNA monomer, " D "=DNA and " P "=peptide.Cochain is a sense strand, and chain is an antisense strand down.
In another this type of embodiment, dsRNA comprises:
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P?YXXXXXXXXXXXXXXXXXXXXXXXXX-5’
Wherein, " X "=RNA, the outstanding domain that " Y " is made up of 1 to 4 RNA monomer, said RNA monomer randomly be 2 '-O-methyl RNA monomer, " D "=DNA and " P "=peptide.Cochain is a sense strand, and chain is an antisense strand down.
In another this type of embodiment, dsRNA comprises:
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-PXXXXXXXXXXXXXXXXXXXXXXXXX-5’
Wherein, " X "=RNA, the outstanding domain that " Y " is made up of 1 to 4 RNA monomer, said RNA monomer randomly be 2 '-O-methyl RNA monomer, " D "=DNA and " P "=peptide.Cochain is a sense strand, and chain is an antisense strand down.
In another this type of embodiment, dsRNA comprises:
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5’
Wherein, " X "=RNA, the outstanding domain that " Y " is made up of 1 to 4 RNA monomer, said RNA monomer randomly be 2 '-O-methyl RNA monomer, " D "=DNA and " P "=peptide.Cochain is a sense strand, and chain is an antisense strand down.
In another this type of embodiment, dsRNA comprises:
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-PYXXXXXXXXXXXXXXXXXXXXXXXXXP-5’
Wherein, " X "=RNA, the outstanding domain that " Y " is made up of 1 to 4 RNA monomer, said RNA monomer randomly be 2 '-O-methyl RNA monomer, " D "=DNA and " P "=peptide.Cochain is a sense strand, and chain is an antisense strand down.
In another this type of embodiment, dsRNA comprises:
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-5’
Wherein, " X "=RNA, the outstanding domain that " Y " is made up of 1 to 4 RNA monomer, said RNA monomer randomly be 2 '-O-methyl RNA monomer, " D "=DNA and " P "=peptide.Cochain is a sense strand, and chain is an antisense strand down.
In another this type of embodiment, dsRNA comprises:
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-PYXXXXXXXXXXXXXXXXXXXXXXXXXP-5’
Wherein, " X "=RNA, the outstanding domain that " Y " is made up of 1 to 4 RNA monomer, said RNA monomer randomly be 2 '-O-methyl RNA monomer, " D "=DNA and " P "=peptide.Cochain is a sense strand, and chain is an antisense strand down.
In another this type of embodiment, dsRNA comprises:
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-5’
Wherein, " X "=RNA, the outstanding domain that " Y " is made up of 1 to 4 RNA monomer, said RNA monomer randomly be 2 '-O-methyl RNA monomer, " D "=DNA and " P "=peptide.Cochain is a sense strand, and chain is an antisense strand down.
In other embodiments, DsiRNA comprises:
5′-XXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-YXXXXXXXXXXXXXXXXXXXXXXXXX?P-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-PYXXXXXXXXXXXXXXXXXXXXXXXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-PXXXXXXXXXXXXXXXXXXXXXXXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXX?P-3′
3′-YXXXXXXXXXXXXXXXXXXXXXXXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-PYXXXXXXXXXXXXXXXXXXXXXXXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-PXXXXXXXXXXXXXXXXXXXXXXXXX?P-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-PYXXXXXXXXXXXXXXXXXXXXXXXXXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′- XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′- XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′- XXXXXXXXXXXXXXXXXXXXXXXXXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′- XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XXXXXXXXXXXXXXXXXXXXXXXXXXXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-P XXXXXXXXXXXXXXXXXXXXXXXXXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′- XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′- XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′- XXXXXXXXXXXXXXXXXXXXXXXXXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′- XXXXXXXXXXXXXXXXXXXXXXXXXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XXXXXXXXXXXXXXXXXXXXXXXXXXXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-P XXXXXXXXXXXXXXXXXXXXXXXXXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-Y XX XX XX XX XX XX XX XXXXXXXXXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-Y XX XX XX XX XX XX XX XXXXXXXXXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-Y XX XX XX XX XX XX XX XXXXXXXXXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-PY XX XX XX XX XX XX XX XXXXXXXXXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XX XX XX XX XX XX XX XXXXXXXXXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-Y XX XX XX XX XX XX XX XXXXXXXXXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-PY XX XX XX XX XX XX XX XXXXXXXXXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XX XX XX XX XX XX XX XXXXXXXXXXX?P-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-PY XX XX XX XX XX XX XX XXXXXXXXXXXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-P XX XX XX XX XX XX XX XXXXXXXXXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′- XXXX XX XX XX XX XX XX XXXXXXXXXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′- XXXX XX XX XX XX XX XX XXXXXXXXXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′- XXXX XX XX XX XX XX XX XXXXXXXXXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XXXX XX XX XX XX XX XX XXXXXXXXXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′- XXXX XX XX XX XX XX XX XXXXXXXXXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XXXX XX XX XX XX XX XX XXXXXXXXXXXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-P XXXX XX XX XX XX XX XX XXXXXXXXXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′- XX XX XX XX XX XX XX XX XXXXXXXXXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′- XX XX XX XX XX XX XX XX XXXXXXXXXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′- XX XX XX XX XX XX XX XX XXXXXXXXXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XX XX XX XX XX XX XX XX XXXXXXXXXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′- XX XX XX XX XX XX XX XX XXXXXXXXXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XX XX XX XX XX XX XX XX XXXXXXXXXXXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-P XX XX XX XX XX XX XX XX XXXXXXXXXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-Y XX XX XX XX XX XX XX XXXXXXXXXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-Y XX XX XX XX XX XX XX XXXXXXXXXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-Y XX XX XX XX XX XX XX XXXXXXXXXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-PY XX XX XX XX XX XX XX XXXXXXXXXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XX XX XX XX XX XX XX XXXXXXXXXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-Y XX XX XX XX XX XX XX XXXXXXXXXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-PY XX XX XX XX XX XX XX XXXXXXXXXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XX XX XX XX XX XX XX XXXXXXXXXXXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-PY XX XX XX XX XX XX XX XXXXXXXXXXXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-P XX XX XX XX XX XX XX XXXXXXXXXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′- XXXX XX XX XX XX XX XX XXXXXXXXXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′- XXXX XX XX XX XX XX XX XXXXXXXXXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′- XXXX XX XX XX XX XX XX XXXXXXXXXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XXXX XX XX XX XX XX XX XXXXXXXXXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′- XXXX XX XX XX XX XX XX XXXXXXXXXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XXXX XX XX XX XX XX XX XXXXXXXXXXXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-P XXXX XX XX XX XX XX XX XXXXXXXXXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′- XX XX XX XX XX XX XX XX XXXXXXXXXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′- XX XX XX XX XX XX XX XX XXXXXXXXXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′- XX XX XX XX XX XX XX XX XXXXXXXXXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XX XX XX XX XX XX XX XX XXXXXXXXXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′- XX XX XX XX XX XX XX XX XXXXXXXXXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XX XX XX XX XX XX XX XX XXXXXXXXXXXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-P XX XX XX XX XX XX XX XX XXXXXXXXXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-Y XX XX XX XX XX XX XX XX XXXXXXXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-Y XX XX XX XX XX XX XX XX XXXXXXXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-Y XX XX XX XX XX XX XX XX XXXXXXXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-PY XX XX XX XX XX XX XX XX XXXXXXXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XX XX XX XX XX XX XX XX XXXXXXXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-Y XX XX XX XX XX XX XX XX XXXXXXXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-PY XX XX XX XX XX XX XX XX XXXXXXXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XX XX XX XX XX XX XX XX XXXXXXXXXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-PY XX XX XX XX XX XX XX XX XXXXXXXXXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-P XX XX XX XX XX XX XX XX XXXXXXXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′- XXXX XX XX XX XX XX XX XX XXXXXXXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′- XXXX XX XX XX XX XX XX XX XXXXXXXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′- XXXX XX XX XX XX XX XX XX XXXXXXXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XXXX XX XX XX XX XX XX XX XXXXXXXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′- XXXX XX XX XX XX XX XX XX XXXXXXXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XXXX XX XX XX XX XX XX XX XXXXXXXXXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-P XXXX XX XX XX XX XX XX XX XXXXXXXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′- XX XX XX XX XX XX XX XX XX XXXXXXXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′- XX XX XX XX XX XX XX XX XX XXXXXXXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′- XX XX XX XX XX XX XX XX XX XXXXXXXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XX XX XX XX XX XX XX XX XX XXXXXXXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′- XX XX XX XX XX XX XX XX XX XXXXXXXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XX XX XX XX XX XX XX XX XX XXXXXXXXXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-P XX XX XX XX XX XX XX XX XX XXXXXXXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-Y XX XX XX XX XX XX XX XX XXXXXXXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-Y XX XX XX XX XX XX XX XX XXXXXXXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-Y XX XX XX XX XX XX XX XX XXXXXXXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-PY XX XX XX XX XX XX XX XX XXXXXXXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XX XX XX XX XX XX XX XX XXXXXXXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-Y XX XX XX XX XX XX XX XX XXXXXXXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-PY XX XX XX XX XX XX XX XX XXXXXXXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XX XX XX XX XX XX XX XX XXXXXXXXXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-PY XX XX XX XX XX XX XX XX XXXXXXXXXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-P XX XX XX XX XX XX XX XX XXXXXXXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′- XXXX XX XX XX XX XX XX XX XXXXXXXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′- XXXX XX XX XX XX XX XX XX XXXXXXXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′- XXXX XX XX XX XX XX XX XX XXXXXXXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XXXX XX XX XX XX XX XX XX XXXXXXXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′- XXXX XX XX XX XX XX XX XX XXXXXXXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XXXX XX XX XX XX XX XX XX XXXXXXXXXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-P XXXX XX XX XX XX XX XX XX XXXXXXXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′- XX XX XX XX XX XX XX XX XX XXXXXXXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′- XX XX XX XX XX XX XX XX XX XXXXXXXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′- XX XX XX XX XX XX XX XX XX XXXXXXXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XX XX XX XX XX XX XX XX XX XXXXXXXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′- XX XX XX XX XX XX XX XX XX XXXXXXXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XX XX XX XX XX XX XX XX XX XXXXXXXXXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-P XX XX XX XX XX XX XX XX XX XXXXXXXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-YXXXXXXXXXXX XX XX XXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-YXXXXXXXXXXX XX XX XXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-YXXXXXXXXXXX XX XX XXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-PYXXXXXXXXXXX XX XX XXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-PXXXXXXXXXXX XX XX XXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-YXXXXXXXXXXX XX XX XXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-PYXXXXXXXXXXX XX XX XXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-PXXXXXXXXXXX XX XX XXXXXXXX XXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-PYXXXXXXXXXXX XX XX XXXXXXXX XXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-PXXXXXXXXXXX XX XX XXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′- XXXXXXXXXXXXX XX XX XXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′- XXXXXXXXXXXXX XX XX XXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′- XXXXXXXXXXXXX XX XX XXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XXXXXXXXXXXXX XX XX XXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′- XXXXXXXXXXXXX XX XX XXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XXXXXXXXXXXXX XX XX XXXXXXXX XXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-P XXXXXXXXXXXXX XX XX XXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′- XXXXXXXXXXXXX XX XX XXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′- XXXXXXXXXXXXX XX XX XXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′- XXXXXXXXXXXXX XX XX XXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XXXXXXXXXXXXX XX XX XXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′- XXXXXXXXXXXXX XX XX XXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XXXXXXXXXXXXX XX XX XXXXXXXX XXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-P XXXXXXXXXXXXX XX XX XXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-YXXXXXXXXXXX XX XX XXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-YXXXXXXXXXXX XX XX XXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-YXXXXXXXXXXX XX XX XXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-PYXXXXXXXXXXX XX XX XXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-PXXXXXXXXXXX XX XX XXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-YXXXXXXXXXXX XX XX XXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-PYXXXXXXXXXXX XX XX XXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-PXXXXXXXXXXX XX XX XXXXXXXX XXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-PYXXXXXXXXXXX XX XX XXXXXXXX XXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-PXXXXXXXXXXX XX XX XXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′- XXXXXXXXXXXXX XX XX XXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′- XXXXXXXXXXXXX XX XX XXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′- XXXXXXXXXXXXX XX XX XXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XXXXXXXXXXXXX XX XX XXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′- XXXXXXXXXXXXX XX XX XXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XXXXXXXXXXXXX XX XX XXXXXXXX XXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-P XXXXXXXXXXXXX XX XX XXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′- XXXXXXXXXXXXX XX XX XXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′- XXXXXXXXXXXXX XX XX XXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′- XXXXXXXXXXXXX XX XX XXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XXXXXXXXXXXXX XX XX XXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′- XXXXXXXXXXXXX XX XX XXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XXXXXXXXXXXXX XX XX XXXXXXXX XXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-P XXXXXXXXXXXXX XX XX XXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-Y XXXXXXXXXXX XX XX XXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-Y XXXXXXXXXXX XX XX XXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-Y XXXXXXXXXXX XX XX XXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-PY XXXXXXXXXXX XX XX XXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XXXXXXXXXXX XX XX XXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-Y XXXXXXXXXXX XX XX XXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-PY XXXXXXXXXXX XX XX XXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XXXXXXXXXXX XX XX XXXXXXXX XXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-PY XXXXXXXXXXX XX XX XXXXXXXX XXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-P XXXXXXXXXXX XX XX XXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′- XXXXXXXXXXXXX XX XX XXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′- XXXXXXXXXXXXX XX XX XXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′- XXXXXXXXXXXXX XX XX XXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XXXXXXXXXXXXX XX XX XXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′- XXXXXXXXXXXXX XX XX XXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XXXXXXXXXXXXX XX XX XXXXXXXX XXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-P XXXXXXXXXXXXX XX XX XXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′- XX XXXXXXXXXXX XX XX XXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′- XX XXXXXXXXXXX XX XX XXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′- XX XXXXXXXXXXX XX XX XXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XX XXXXXXXXXXX XX XX XXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′- XX XXXXXXXXXXX XX XX XXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XX XXXXXXXXXXX XX XX XXXXXXXX XXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-P XX XXXXXXXXXXX XX XX XXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-Y XXXXXXXXXXX XX XX XXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-Y XXXXXXXXXXX XX XX XXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-Y XXXXXXXXXXX XX XX XXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-PY XXXXXXXXXXX XX XX XXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XXXXXXXXXXX XX XX XXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-Y XXXXXXXXXXX XX XX XXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-PY XXXXXXXXXXX XX XX XXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XXXXXXXXXXX XX XX XXXXXXXX XXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-PY XXXXXXXXXXX XX XX XXXXXXXX XXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXX?P-3′
3′-P XXXXXXXXXXX XX XX XXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′- XXXXXXXXXXXXX XX XX XXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′- XXXXXXXXXXXXX XX XX XXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′- XXXXXXXXXXXXX XX XX XXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XXXXXXXXXXXXX XX XX XXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′- XXXXXXXXXXXXX XX XX XXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XXXXXXXXXXXXX XX XX XXXXXXXX XXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-P XXXXXXXXXXXXX XX XX XXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′- XX XXXXXXXXXXX XX XX XXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′- XX XXXXXXXXXXX XX XX XXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′- XX XXXXXXXXXXX XX XX XXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XX XXXXXXXXXXX XX XX XXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′- XX XXXXXXXXXXX XX XX XXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XX XXXXXXXXXXX XX XX XXXXXXXX XXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-P XX XXXXXXXXXXX XX XX XXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-Y XX XXXXXXXXX XX XX XXXXXXXXX X-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-Y XX XXXXXXXXX XX XX XXXXXXXXX X-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-Y XX XXXXXXXXX XX XX XXXXXXXXX XP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-PY XX XXXXXXXXX XX XX XXXXXXXXX X-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XX XXXXXXXXX XX XX XXXXXXXXX X-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-Y XX XXXXXXXXX XX XX XXXXXXXXX X-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-PY XX XXXXXXXXX XX XX XXXXXXXXX XP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XX XXXXXXXXX XX XX XXXXXXXXX XP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-PY XX XXXXXXXXX XX XX XXXXXXXXX XP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-P XX XXXXXXXXX XX XX XXXXXXXXX XP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′- XXXX XXXXXXXXX XX XX XXXXXXXXX X-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′- XXXX XXXXXXXXX XX XX XXXXXXXXX X-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′- XXXX XXXXXXXXX XX XX XXXXXXXXX XP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XXXX XXXXXXXXX XX XX XXXXXXXXX X-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′- XXXX XXXXXXXXX XX XX XXXXXXXXX X-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XXXX XXXXXXXXX XX XX XXXXXXXXX XP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-P XXXX XXXXXXXXX XX XX XXXXXXXXX XP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′- XX XX XXXXXXXXX XX XX XXXXXXXXX X-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′- XX XX XXXXXXXXX XX XX XXXXXXXXX X-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′- XX XX XXXXXXXXX XX XX XXXXXXXXX XP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XX XX XXXXXXXXX XX XX XXXXXXXXX X-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′- XX XX XXXXXXXXX XX XX XXXXXXXXX X-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XX XX XXXXXXXXX XX XX XXXXXXXXX X?P-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-P XX XX XXXXXXXXX XX XX XXXXXXXXX X?P-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-Y XX XXXXXXXXX XX XX XXXXXXXXX X-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-Y XX XXXXXXXXX XX XX XXXXXXXXX X-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-Y XX XXXXXXXXX XX XX XXXXXXXXX XP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-PY XX XXXXXXXXX XX XX XXXXXXXXX X-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XX XXXXXXXXX XX XX XXXXXXXXX X-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-Y XX XXXXXXXXX XX XX XXXXXXXXX X-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-PY XX XXXXXXXXX XX XX XXXXXXXXX XP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XX XXXXXXXXX XX XX XXXXXXXXX XP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-PY XX XXXXXXXXX XX XX XXXXXXXXX XP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-P XX XXXXXXXXX XX XX XXXXXXXXX XP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′- XXXX XXXXXXXXX XX XX XXXXXXXXX X-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′- XXXX XXXXXXXXX XX XX XXXXXXXXX X-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′- XXXX XXXXXXXXX XX XX XXXXXXXXX XP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XXXX XXXXXXXXX XX XX XXXXXXXXX X-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′- XXXX XXXXXXXXX XX XX XXXXXXXXX X-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XXXX XXXXXXXXX XX XX XXXXXXXXX XP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-P XXXX XXXXXXXXX XX XX XXXXXXXXX XP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′- XX XX XXXXXXXXX XX XX XXXXXXXXX X-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′- XX XX XXXXXXXXX XX XX XXXXXXXXX X-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′- XX XX XXXXXXXXX XX XX XXXXXXXXX XP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XX XX XXXXXXXXX XX XX XXXXXXXXX X-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′- XX XX XXXXXXXXX XX XX XXXXXXXXX X-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XX XX XXXXXXXXX XX XX XXXXXXXXX XP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-P XX XX XXXXXXXXX XX XX XXXXXXXXX XP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-Y XX XX XXXXXXX XX XX XXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-Y XX XX XXXXXXX XX XX XXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-Y XX XX XXXXXXX XX XX XXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-PY XX XX XXXXXXX XX XX XXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XX XX XXXXXXX XX XX XXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-Y XX XX XXXXXXX XX XX XXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-PY XX XX XXXXXXX XX XX XXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XX XX XXXXXXX XX XX XXXXXXXX XXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-PY XX XX XXXXXXX XX XX XXXXXXXX XXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-P XX XX XXXXXXX XX XX XXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′- XXXX XX XXXXXXX XX XX XXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′- XXXX XX XXXXXXX XX XX XXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′- XXXX XX XXXXXXX XX XX XXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XXXX XX XXXXXXX XX XX XXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′- XXXX XX XXXXXXX XX XX XXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XXXX XX XXXXXXX XX XX XXXXXXXX XXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-P XXXX XX XXXXXXX XX XX XXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′- XX XX XX XXXXXXX XX XX XXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′- XX XX XX XXXXXXX XX XX XXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′- XX XX XX XXXXXXX XX XX XXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XX XX XX XXXXXXX XX XX XXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′- XX XX XX XXXXXXX XX XX XXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XX XX XX XXXXXXX XX XX XXXXXXXX XXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-P XX XX XX XXXXXXX XX XX XXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-Y XX XX XXXXXXX XX XX XXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-Y XX XX XXXXXXX XX XX XXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-Y XX XX XXXXXXX XX XX XXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-PY XX XX XXXXXXX XX XX XXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XX XX XXXXXXX XX XX XXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-Y XX XX XXXXXXX XX XX XXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-PY XX XX XXXXXXX XX XX XXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XX XX XXXXXXX XX XX XXXXXXXX XXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-PY XX XX XXXXXXX XX XX XXXXXXXX XXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-P XX XX XXXXXXX XX XX XXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′- XXXX XX XXXXXXX XX XX XXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′- XXXX XX XXXXXXX XX XX XXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′- XXXX XX XXXXXXX XX XX XXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XXXX XX XXXXXXX XX XX XXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′- XXXX XX XXXXXXX XX XX XXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XXXX XX XXXXXXX XX XX XXXXXXXX XXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-P XXXX XX XXXXXXX XX XX XXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′- XX XX XX XXXXXXX XX XX XXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′- XX XX XX XXXXXXX XX XX XXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′- XX XX XX XXXXXXX XX XX XXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XX XX XX XXXXXXX XX XX XXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′- XX XX XX XXXXXXX XX XX XXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XX XX XX XXXXXXX XX XX XXXXXXXX XXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-P XX XX XX XXXXXXX XX XX XXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-Y XX XX XXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-Y XX XX XXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-Y XX XX XXXXXXXX XX XXXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-PY XX XX XXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XX XX XXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-Y XX XX XXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-PY XX XX XXXXXXXX XX XXXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XX XX XXXXXXXX XX XXXXXXX XXXXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-PY XX XX XXXXXXXX XX XXXXXXX XXXXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-P XX XX XXXXXXXX XX XXXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′- XXXX XX XXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′- XXXX XX XXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′- XXXX XX XXXXXXXX XX XXXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XXXX XX XXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′- XXXX XX XXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XXXX XX XXXXXXXX XX XXXXXXX XXXX?P-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-P XXXX XX XXXXXXXX XX XXXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′- XX XX XX XXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′- XX XX XX XXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′- XX XX XX XXXXXXXX XX XXXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XX XX XX XXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′- XX XX XX XXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XX XX XX XXXXXXXX XX XXXXXXX XXXXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-P XX XX XX XXXXXXXX XX XXXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-Y XX XX XXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-Y XX XX XXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-Y XX XX XXXXXXXX XX XXXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-PY XX XX XXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XX XX XXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-Y XX XX XXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-PY XX XX XXXXXXXX XX XXXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XX XX XXXXXXXX XX XXXXXXX XXXXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-PY XX XX XXXXXXXX XX XXXXXXX XXXXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-P XX XX XXXXXXXX XX XXXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′- XXXX XX XXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′- XXXX XX XXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′- XXXX XX XXXXXXXX XX XXXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XXXX XX XXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′- XXXX XX XXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XXXX XX XXXXXXXX XX XXXXXXX XXXXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-P XXXX XX XXXXXXXX XX XXXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′- XX XX XX XXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′- XX XX XX XXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′- XX XX XX XXXXXXXX XX XXXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XX XX XX XXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′- XX XX XX XXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XX XX XX XXXXXXXX XX XXXXXXX XXXXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXp-3′
3′-P XX XX XX XXXXXXXX XX XXXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-Y XX XX XXXXXXXXXXXXXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-Y XX XX XXXXXXXXXXXXXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-Y XX XX XXXXXXXXXXXXXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-PY XX XX XXXXXXXXXXXXXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XX XX XXXXXXXXXXXXXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-Y XX XX XXXXXXXXXXXXXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-PY XX XX XXXXXXXXXXXXXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XX XX XXXXXXXXXXXXXXXXX XXXXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-PY XX XX XXXXXXXXXXXXXXXXX XXXXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-P XX XX XXXXXXXXXXXXXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′- XXXX XX XXXXXXXXXXXXXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′- XXXX XX XXXXXXXXXXXXXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′- XXXX XX XXXXXXXXXXXXXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XXXX XX XXXXXXXXXXXXXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′- XXXX XX XXXXXXXXXXXXXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XXXX XX XXXXXXXXXXXXXXXXX XXXXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-P XXXX XX XXXXXXXXXXXXXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′- XX XX XX XXXXXXXXXXXXXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′- XX XX XX XXXXXXXXXXXXXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′- XX XX XX XXXXXXXXXXXXXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XX XX XX XXXXXXXXXXXXXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′- XX XX XX XXXXXXXXXXXXXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XX XX XX XXXXXXXXXXXXXXXXX XXXXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-P XX XX XX XXXXXXXXXXXXXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-Y XX XX XXXXXXXXXXXXXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-Y XX XX XXXXXXXXXXXXXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-Y XX XX XXXXXXXXXXXXXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-PY XX XX XXXXXXXXXXXXXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XX XX XXXXXXXXXXXXXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-Y XX XX XXXXXXXXXXXXXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-PY XX XX XXXXXXXXXXXXXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XX XX XXXXXXXXXXXXXXXXX XXXXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-PY XX XX XXXXXXXXXXXXXXXXX XXXXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-P XX XX XXXXXXXXXXXXXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′- XXXX XX XXXXXXXXXXXXXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′- XXXX XX XXXXXXXXXXXXXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′- XXXX XX XXXXXXXXXXXXXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XXXX XX XXXXXXXXXXXXXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′- XXXX XX XXXXXXXXXXXXXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XXXX XX XXXXXXXXXXXXXXXXX XXXXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-P XXXX XX XXXXXXXXXXXXXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′- XX XX XX XXXXXXXXXXXXXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′- XX XX XX XXXXXXXXXXXXXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′- XX XX XX XXXXXXXXXXXXXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XX XX XX XXXXXXXXXXXXXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′- XX XX XX XXXXXXXXXXXXXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XX XX XX XXXXXXXXXXXXXXXXX XXXXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-P XX XX XX XXXXXXXXXXXXXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-Y XX XX XXXXXX XXX XX XXXXXXXXX X-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-Y XX XX XXXXXX XXX XX XXXXXXXXX X-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-Y XX XX XXXXXX XXX XX XXXXXXXXX XP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-PY XX XX XXXXXX XXX XX XXXXXXXXX X-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XX XX XXXXXX XXX XX XXXXXXXXX X-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-Y XX XX XXXXXX XXX XX XXXXXXXXX X-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-PY XX XX XXXXXX XXX XX XXXXXXXXX XP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XX XX XXXXXX XXX XX XXXXXXXXX XP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-PY XX XX XXXXXX XXX XX XXXXXXXXX XP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-P XX XX XXXXXX XXX XX XXXXXXXXX XP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′- XXXX XX XXXXXX XXX XX XXXXXXXXX X-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′- XXXX XX XXXXXX XXX XX XXXXXXXXX X-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′- XXXX XX XXXXXX XXX XX XXXXXXXXX XP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XXXX XX XXXXXX XXX XX XXXXXXXXX X-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′- XXXX XX XXXXXX XXX XX XXXXXXXXX X-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XXXX XX XXXXXX XXX XX XXXXXXXXX XP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-P XXXX XX XXXXXX XXX XX XXXXXXXXX XP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′- XX XX XX XXXXXX XXX XX XXXXXXXXX X-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′- XX XX XX XXXXXX XXX XX XXXXXXXXX X-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′- XX XX XX XXXXXX XXX XX XXXXXXXXX XP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XX XX XX XXXXXX XXX XX XXXXXXXXX X-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′- XX XX XX XXXXXX XXX XX XXXXXXXXX X-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XX XX XX XXXXXX XXX XX XXXXXXXXX XP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-P XX XX XX XXXXXX XXX XX XXXXXXXXX XP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-Y XX XX XXXXXX XXX XX XXXXXXXXX X-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-Y XX XX XXXXXX XXX XX XXXXXXXXX X-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-Y XX XX XXXXXX XXX XX XXXXXXXXX XP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-PY XX XX XXXXXX XXX XX XXXXXXXXX X-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XX XX XXXXXX XXX XX XXXXXXXXX X-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-Y XX XX XXXXXX XXX XX XXXXXXXXX X-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-PY XX XX XXXXXX XXX XX XXXXXXXXX XP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XX XX XXXXXX XXX XX XXXXXXXXX XP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-PY XX XX XXXXXX XXX XX XXXXXXXXX XP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-P XX XX XXXXXX XXX XX XXXXXXXXX XP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′- XXXX XX XXXXXX XXX XX XXXXXXXXX X-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′- XXXX XX XXXXXX XXX XX XXXXXXXXX X-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′- XXXX XX XXXXXX XXX XX XXXXXXXXX XP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XXXX XX XXXXXX XXX XX XXXXXXXXX X-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′- XXXX XX XXXXXX XXX XX XXXXXXXXX X-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XXXX XX XXXXXX XXX XX XXXXXXXXX XP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-P XXXX XX XXXXXX XXX XX XXXXXXXXX XP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′- XX XX XX XXXXXX XXX XX XXXXXXXXX X-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′- XX XX XX XXXXXX XXX XX XXXXXXXXX X-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′- XX XX XX XXXXXX XXX XX XXXXXXXXX XP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XX XX XX XXXXXX XXX XX XXXXXXXXX X-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′- XX XX XX XXXXXX XXX XX XXXXXXXXX X-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XX XX XX XXXXXX XXX XX XXXXXXXXX XP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-P XX XX XX XXXXXX XXX XX XXXXXXXXX XP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-Y XX XX XXXXXX XXX XX XXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-Y XX XX XXXXXX XXX XX XXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-Y XX XX XXXXXX XXX XX XXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-PY XX XX XXXXXX XXX XX XXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XX XX XXXXXX XXX XX XXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-Y XX XX XXXXXX XXX XX XXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-PY XX XX XXXXXX XXX XX XXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XX XX XXXXXX XXX XX XXXXXX XXXXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-PY XX XX XXXXXX XXX XX XXXXXX XXXXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-P XX XX XXXXXX XXX XX XXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′- XXXX XX XXXXXX XXX XX XXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′- XXXX XX XXXXXX XXX XX XXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′- XXXX XX XXXXXX XXX XX XXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XXXX XX XXXXXX XXX XX XXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′- XXXX XX XXXXXX XXX XX XXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XXXX XX XXXXXX XXX XX XXXXXX XXXX?P-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-P XXXX XX XXXXXX XXX XX XXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′- XX XX XX XXXXXX XXX XX XXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′- XX XX XX XXXXXX XXX XX XXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′- XX XX XX XXXXXX XXX XX XXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XX XX XX XXXXXX XXX XX XXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′- XX XX XX XXXXXX XXX XX XXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XX XX XX XXXXXX XXX XX XXXXXX XXXXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-P XX XX XX XXXXXX XXX XX XXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-Y XX XX XXXXXX XXX XX XXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-Y XX XX XXXXXX XXX XX XXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-Y XX XX XXXXXX XXX XX XXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-PY XX XX XXXXXX XXX XX XXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XX XX XXXXXX XXX XX XXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-Y XX XX XXXXXX XXX XX XXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-PY XX XX XXXXXX XXX XX XXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XX XX XXXXXX XXX XX XXXXXX XXXXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-PY XX XX XXXXXX XXX XX XXXXXX XXXXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-P XX XX XXXXXX XXX XX XXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′- XXXX XX XXXXXX XXX XX XXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′- XXXX XX XXXXXX XXX XX XXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′- XXXX XX XXXXXX XXX XX XXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XXXX XX XXXXXX XXX XX XXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′- XXXX XX XXXXXX XXX XX XXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XXXX XX XXXXXX XXX XX XXXXXX XXXXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-P XXXX XX XXXXXX XXX XX XXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′- XX XX XX XXXXXX XXX XX XXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′- XX XX XX XXXXXX XXX XX XXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′- XX XX XX XXXXXX XXX XX XXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XX XX XX XXXXXX XXX XX XXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′- XX XX XX XXXXXX XXX XX XXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XX XX XX XXXXXX XXX XX XXXXXX XXXXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-P XX XX XX XXXXXX XXX XX XXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-YX XX XXXXXX XXXX XXXXXXXXXXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-YX XX XXXXXX XXXX XXXXXXXXXXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-YX XX XXXXXX XXXX XXXXXXXXXXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-PYX XX XXXXXX XXXX XXXXXXXXXXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-PX XX XXXXXX XXXX XXXXXXXXXXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-YX XX XXXXXX XXXX XXXXXXXXXXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-PYX XX XXXXXX XXXX XXXXXXXXXXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-PX XX XXXXXX XXXX XXXXXXXXXXXXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-PYX XX XXXXXX XXXX XXXXXXXXXXXXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-PX XX XXXXXX XXXX XXXXXXXXXXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′- XXX XX XXXXXX XXXX XXXXXXXXXXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′- XXX XX XXXXXX XXXX XXXXXXXXXXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′- XXX XX XXXXXX XXXX XXXXXXXXXXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XXX XX XXXXXX XXXX XXXXXXXXXXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′- XXX XX XXXXXX XXXX XXXXXXXXXXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XXX XX XXXXXX XXXX XXXXXXXXXXXXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-P XXX XX XXXXXX XXXX XXXXXXXXXXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′- XXX XX XXXXXX XXXX XXXXXXXXXXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′- XXX XX XXXXXX XXXX XXXXXXXXXXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′- XXX XX XXXXXX XXXX XXXXXXXXXXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XXX XX XXXXXX XXXX XXXXXXXXXXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′- XXX XX XXXXXX XXXX XXXXXXXXXXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XXX XX XXXXXX XXXX XXXXXXXXXXXXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-P XXX XX XXXXXX XXXX XXXXXXXXXXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-YX XX XXXXXX XXXX XXXXXXXXXXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-YX XX XXXXXX XXXX XXXXXXXXXXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-YX XX XXXXXX XXXX XXXXXXXXXXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-PYX XX XXXXXX XXXX XXXXXXXXXXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-PX XX XXXXXX XXXX XXXXXXXXXXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-YX XX XXXXXX XXXX XXXXXXXXXXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-PYX XX XXXXXX XXXX XXXXXXXXXXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-PX XX XXXXXX XXXX XXXXXXXXXXXXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-PYX XX XXXXXX XXXX XXXXXXXXXXXXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-PX XX XXXXXX XXXX XXXXXXXXXXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′- XXX XX XXXXXX XXXX XXXXXXXXXXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′- XXX XX XXXXXX XXXX XXXXXXXXXXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′- XXX XX XXXXXX XXXX XXXXXXXXXXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XXX XX XXXXXX XXXX XXXXXXXXXXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′- XXX XX XXXXXX XXXX XXXXXXXXXXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XXX XX XXXXXX XXXX XXXXXXXXXXXXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-P XXX XX XXXXXX XXXX XXXXXXXXXXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′- XXX XX XXXXXX XXXX XXXXXXXXXXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′- XXX XX XXXXXX XXXX XXXXXXXXXXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′- XXX XX XXXXXX XXXX XXXXXXXXXXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XXX XX XXXXXX XXXX XXXXXXXXXXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′- XXX XX XXXXXX XXXX XXXXXXXXXXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XXX XX XXXXXX XXXX XXXXXXXXXXXXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-P XXX XX XXXXXX XXXX XXXXXXXXXXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-YXX XXX XXXXXXXXXX XXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-YXX XXX XXXXXXXXXX XXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-YXX XXX XXXXXXXXXX XXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-PYXX XXX XXXXXXXXXX XXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-PXX XXX XXXXXXXXXX XXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-YXX XXX XXXXXXXXXX XXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-PYXX XXX XXXXXXXXXX XXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-PXX XXX XXXXXXXXXX XXXXXX XXXXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-PYXX XXX XXXXXXXXXX XXXXXX XXXXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-PXX XXX XXXXXXXXXX XXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′- XXXX XXX XXXXXXXXXX XXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′- XXXX XXX XXXXXXXXXX XXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′- XXXX XXX XXXXXXXXXX XXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XXXX XXX XXXXXXXXXX XXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′- XXXX XXX XXXXXXXXXX XXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XXXX XXX XXXXXXXXXX XXXXXX XXXXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-P XXXX XXX XXXXXXXXXX XXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′- XXXX XXX XXXXXXXXXX XXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′- XXXX XXX XXXXXXXXXX XXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′- XXXX XXX XXXXXXXXXX XXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XXXX XXX XXXXXXXXXX XXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′- XXXX XXX XXXXXXXXXX XXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XXXX XXX XXXXXXXXXX XXXXXX XXXXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-P XXXX XXX XXXXXXXXXX XXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-YXX XXX XXXXXXXXXX XXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-YXX XXX XXXXXXXXXX XXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-YXX XXX XXXXXXXXXX XXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-PYXX XXX XXXXXXXXXX XXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-PXX XXX XXXXXXXXXX XXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-YXX XXX XXXXXXXXXX XXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-PYXX XXX XXXXXXXXXX XXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-PXX XXX XXXXXXXXXX XXXXXX XXXXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-PYXX XXX XXXXXXXXXX XXXXXX XXXXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-PXX XXX XXXXXXXXXX XXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′- XXXX XXX XXXXXXXXXX XXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′- XXXX XXX XXXXXXXXXX XXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′- XXXX XXX XXXXXXXXXX XXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XXXX XXX XXXXXXXXXX XXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′- XXXX XXX XXXXXXXXXX XXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XXXX XXX XXXXXXXXXX XXXXXX XXXXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-P XXXX XXX XXXXXXXXXX XXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′- XXXX XXX XXXXXXXXXX XXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′- XXXX XXX XXXXXXXXXX XXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′- XXXX XXX XXXXXXXXXX XXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XXXX XXX XXXXXXXXXX XXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′- XXXX XXX XXXXXXXXXX XXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XXXX XXX XXXXXXXXXX XXXXXX XXXXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-P XXXX XXX XXXXXXXXXX XXXXXX XXXXp-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-Y XXXXXXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-Y XXXXXXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-Y XXXXXXXXXXXX XX XXXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-PY XXXXXXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XXXXXXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-Y XXXXXXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-PY XXXXXXXXXXXX XX XXXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XXXXXXXXXXXX XX XXXXXXX XXXXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-PY XXXXXXXXXXXX XX XXXXXXX XXXXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-P XXXXXXXXXXXX XX XXXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′- XXXXXXXXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′- XXXXXXXXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′- XXXXXXXXXXXXXX XX XXXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XXXXXXXXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′- XXXXXXXXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XXXXXXXXXXXXXX XX XXXXXXX XXXXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-P XXXXXXXXXXXXXX XX XXXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′- XX XXXXXXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′- XX XXXXXXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′- XX XXXXXXXXXXXX XX XXXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XX XXXXXXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′- XX XXXXXXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XX XXXXXXXXXXXX XX XXXXXXX XXXXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-P XX XXXXXXXXXXXX XX XXXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-Y XXXXXXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-Y XXXXXXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-Y XXXXXXXXXXXX XX XXXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-PY XXXXXXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XXXXXXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-Y XXXXXXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-PY XXXXXXXXXXXX XX XXXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XXXXXXXXXXXX XX XXXXXXX XXXXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-PY XXXXXXXXXXXX XX XXXXXXX XXXXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-P XXXXXXXXXXXX XX XXXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′- XXXXXXXXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′- XXXXXXXXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′- XXXXXXXXXXXXXX XX XXXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XXXXXXXXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′- XXXXXXXXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XXXXXXXXXXXXXX XX XXXXXXX XXXXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-P XXXXXXXXXXXXXX XX XXXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′- XX XXXXXXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′- XX XXXXXXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′- XX XXXXXXXXXXXX XX XXXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XX XXXXXXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′- XX XXXXXXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XX XXXXXXXXXXXX XX XXXXXXX XXXXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-P XX XXXXXXXXXXXX XX XXXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-Y XX XXXXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-Y XX XXXXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-Y XX XXXXXXXXXX XX XXXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-PY XX XXXXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XX XXXXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-Y XX XXXXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-PY XX XXXXXXXXXX XX XXXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XX XXXXXXXXXX XX XXXXXXX XXXXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-PY XX XXXXXXXXXX XX XXXXXXX XXXXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-P XX XXXXXXXXXX XX XXXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′- XXXX XXXXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′- XXXX XXXXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′- XXXX XXXXXXXXXX XX XXXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XXXX XXXXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′- XXXX XXXXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XXXX XXXXXXXXXX XX XXXXXXX XXXXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-P XXXX XXXXXXXXXX XX XXXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′- XX XX XXXXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′- XX XX XXXXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′- XX XX XXXXXXXXXX XX XXXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XX XX XXXXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′- XX XX XXXXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XX XX XXXXXXXXXX XX XXXXXXX XXXXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-P XX XX XXXXXXXXXX XX XXXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-Y XX XXXXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-Y XX XXXXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-Y XX XXXXXXXXXX XX XXXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-PY XX XXXXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XX XXXXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-Y XX XXXXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-PY XX XXXXXXXXXX XX XXXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XX XXXXXXXXXX XX XXXXXXX XXXXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-PY XX XXXXXXXXXX XX XXXXXXX XXXXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-P XX XXXXXXXXXX XX XXXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′- XXXX XXXXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′- XXXX XXXXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′- XXXX XXXXXXXXXX XX XXXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XXXX XXXXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′- XXXX XXXXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XXXX XXXXXXXXXX XX XXXXXXX XXXXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-P XXXX XXXXXXXXXX XX XXXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′- XX XX XXXXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′- XX XX XXXXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′- XX XX XXXXXXXXXX XX XXXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XX XX XXXXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXX?P-3′
3′- XX XX XXXXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XX XX XXXXXXXXXX XX XXXXXXX XXXXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-P XX XX XXXXXXXXXX XX XXXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-YXX XXXXXXXXXXX XXXXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-YXX XXXXXXXXXXX XXXXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-YXX XXXXXXXXXXX XXXXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-PYXX XXXXXXXXXXX XXXXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-PXX XXXXXXXXXXX XXXXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-YXX XXXXXXXXXXX XXXXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-PYXX XXXXXXXXXXX XXXXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-PXX XXXXXXXXXXX XXXXXXXXXX XXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-PYXX XXXXXXXXXXX XXXXXXXXXX XXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-PXX XXXXXXXXXXX XXXXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′- XXXX XXXXXXXXXXX XXXXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′- XXXX XXXXXXXXXXX XXXXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′- XXXX XXXXXXXXXXX XXXXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XXXX XXXXXXXXXXX XXXXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′- XXXX XXXXXXXXXXX XXXXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XXXX XXXXXXXXXXX XXXXXXXXXX XXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-P XXXX XXXXXXXXXXX XXXXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′- XXXX XXXXXXXXXXX XXXXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′- XXXX XXXXXXXXXXX XXXXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′- XXXX XXXXXXXXXXX XXXXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XXXX XXXXXXXXXXX XXXXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′- XXXX XXXXXXXXXXX XXXXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XXXX XXXXXXXXXXX XXXXXXXXXX XXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-P XXXX XXXXXXXXXXX XXXXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-YXX XXXXXXXXXXX XXXXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-YXX XXXXXXXXXXX XXXXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-YXX XXXXXXXXXXX XXXXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-PYXX XXXXXXXXXXX XXXXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-PXX XXXXXXXXXXX XXXXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-YXX XXXXXXXXXXX XXXXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-PYXX XXXXXXXXXXX XXXXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-PXX XXXXXXXXXXX XXXXXXXXXX XXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-PYXX XXXXXXXXXXX XXXXXXXXXX XXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-PXX XXXXXXXXXXX XXXXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′- XXXX XXXXXXXXXXX XXXXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′- XXXX XXXXXXXXXXX XXXXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′- XXXX XXXXXXXXXXX XXXXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XXXX XXXXXXXXXXX XXXXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′- XXXX XXXXXXXXXXX XXXXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XXXX XXXXXXXXXXX XXXXXXXXXX XXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-P XXXX XXXXXXXXXXX XXXXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′- XXXX XXXXXXXXXXX XXXXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′- XXXX XXXXXXXXXXX XXXXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′- XXXX XXXXXXXXXXX XXXXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XXXX XXXXXXXXXXX XXXXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXX?P-3′
3′- XXXX XXXXXXXXXXX XXXXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XXXX XXXXXXXXXXX XXXXXXXXXX XXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-P XXXX XXXXXXXXXXX XXXXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-Y XXXXXXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-Y XXXXXXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-Y XXXXXXXXXXXX XX XXXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-PY XXXXXXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XXXXXXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-Y XXXXXXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-PY XXXXXXXXXXXX XX XXXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XXXXXXXXXXXX XX XXXXXXX XXXXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-PY XXXXXXXXXXXX XX XXXXXXX XXXXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-P XXXXXXXXXXXX XX XXXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′- XXXXXXXXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′- XXXXXXXXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′- XXXXXXXXXXXXXX XX XXXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XXXXXXXXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′- XXXXXXXXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XXXXXXXXXXXXXX XX XXXXXXX XXXXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-P XXXXXXXXXXXXXX XX XXXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′- XX XXXXXXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′- XX XXXXXXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′- XX XXXXXXXXXXXX XX XXXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XX XXXXXXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′- XX XXXXXXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XX XXXXXXXXXXXX XX XXXXXXX XXXXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-P XX XXXXXXXXXXXX XX XXXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-Y XXXXXXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-Y XXXXXXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-Y XXXXXXXXXXXX XX XXXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-PY XXXXXXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XXXXXXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-Y XXXXXXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-PY XXXXXXXXXXXX XX XXXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XXXXXXXXXXXX XX XXXXXXX XXXXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-PY XXXXXXXXXXXX XX XXXXXXX XXXXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-P XXXXXXXXXXXX XX XXXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′- XXXXXXXXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′- XXXXXXXXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′- XXXXXXXXXXXXXX XX XXXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XXXXXXXXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′- XXXXXXXXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XXXXXXXXXXXXXX XX XXXXXXX XXXXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-P XXXXXXXXXXXXXX XX XXXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′- XX XXXXXXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′- XX XXXXXXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′- XX XXXXXXXXXXXX XX XXXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XX XXXXXXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′- XX XXXXXXXXXXXX XX XXXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XX XXXXXXXXXXXX XX XXXXXXX XXXXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-P XX XXXXXXXXXXXX XX XXXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-YXX XXX XXXXXXXX XXXXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-YXX XXX XXXXXXXX XXXXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-YXX XXX XXXXXXXX XXXXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-PYXX XXX XXXXXXXX XXXXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-PXX XXX XXXXXXXX XXXXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-YXX XXX XXXXXXXX XXXXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-PYXX XXX XXXXXXXX XXXXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-PXX XXX XXXXXXXX XXXXXXXXXX XXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-PYXX XXX XXXXXXXX XXXXXXXXXX XXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-PXX XXX XXXXXXXX XXXXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′- XXXX XXX XXXXXXXX XXXXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′- XXXX XXX XXXXXXXX XXXXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′- XXXX XXX XXXXXXXX XXXXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XXXX XXX XXXXXXXX XXXXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′- XXXX XXX XXXXXXXX XXXXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XXXX XXX XXXXXXXX XXXXXXXXXX XXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-P XXXX XXX XXXXXXXX XXXXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′- XXXX XXX XXXXXXXX XXXXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′- XXXX XXX XXXXXXXX XXXXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′- XXXX XXX XXXXXXXX XXXXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XXXX XXX XXXXXXXX XXXXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′- XXXX XXX XXXXXXXX XXXXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XXXX XXX XXXXXXXX XXXXXXXXXX XXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-P XXXX XXX XXXXXXXX XXXXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-YXX XXX XXXXXXXX XXXXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-YXX XXX XXXXXXXX XXXXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-YXX XXX XXXXXXXX XXXXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-PYXX XXX XXXXXXXX XXXXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-PXX XXX XXXXXXXX XXXXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-YXX XXX XXXXXXXX XXXXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-PYXX XXX XXXXXXXX XXXXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-PXX XXX XXXXXXXX XXXXXXXXXX XXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-PYXX XXX XXXXXXXX XXXXXXXXXX XXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-PXX XXX XXXXXXXX XXXXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′- XXXX XXX XXXXXXXX XXXXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′- XXXX XXX XXXXXXXX XXXXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′- XXXX XXX XXXXXXXX XXXXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XXXX XXX XXXXXXXX XXXXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′- XXXX XXX XXXXXXXX XXXXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XXXX XXX XXXXXXXX XXXXXXXXXX XXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-P XXXX XXX XXXXXXXX XXXXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′- XXXX XXX XXXXXXXX XXXXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′- XXXX XXX XXXXXXXX XXXXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′- XXXX XXX XXXXXXXX XXXXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XXXX XXX XXXXXXXX XXXXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′- XXXX XXX XXXXXXXX XXXXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XXXX XXX XXXXXXXX XXXXXXXXXX XXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-P XXXX XXX XXXXXXXX XXXXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-YXXXXXXXXXXXXX XXXXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-YXXXXXXXXXXXXX XXXXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-YXXXXXXXXXXXXX XXXXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-PYXXXXXXXXXXXXX XXXXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-PXXXXXXXXXXXXX XXXXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-YXXXXXXXXXXXXX XXXXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-PYXXXXXXXXXXXXX XXXXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-PXXXXXXXXXXXXX XXXXXXXXXX XXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-PYXXXXXXXXXXXXX XXXXXXXXXX XXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-PXXXXXXXXXXXXX XXXXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′- XXXXXXXXXXXXXXX XXXXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′- XXXXXXXXXXXXXXX XXXXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′- XXXXXXXXXXXXXXX XXXXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XXXXXXXXXXXXXXX XXXXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′- XXXXXXXXXXXXXXX XXXXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XXXXXXXXXXXXXXX XXXXXXXXXX XXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-P XXXXXXXXXXXXXXX XXXXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′- XXXXXXXXXXXXXXX XXXXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′- XXXXXXXXXXXXXXX XXXXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′- XXXXXXXXXXXXXXX XXXXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XXXXXXXXXXXXXXX XXXXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′- XXXXXXXXXXXXXXX XXXXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XXXXXXXXXXXXXXX XXXXXXXXXX XXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-P XXXXXXXXXXXXXXX XXXXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-YXXXXXXXXXXXXX XXXXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-YXXXXXXXXXXXXX XXXXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-YXXXXXXXXXXXXX XXXXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-PYXXXXXXXXXXXXX XXXXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-PXXXXXXXXXXXXX XXXXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-YXXXXXXXXXXXXX XXXXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-PYXXXXXXXXXXXXX XXXXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-PXXXXXXXXXXXXX XXXXXXXXXX XXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-PYXXXXXXXXXXXXX XXXXXXXXXX XXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-PXXXXXXXXXXXXX XXXXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′- XXXXXXXXXXXXXXX XXXXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′- XXXXXXXXXXXXXXX XXXXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′- XXXXXXXXXXXXXXX XXXXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XXXXXXXXXXXXXXX XXXXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′- XXXXXXXXXXXXXXX XXXXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XXXXXXXXXXXXXXX XXXXXXXXXX XXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-P XXXXXXXXXXXXXXX XXXXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′- XXXXXXXXXXXXXXX XXXXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′- XXXXXXXXXXXXXXX XXXXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′- XXXXXXXXXXXXXXX XXXXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XXXXXXXXXXXXXXX XXXXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′- XXXXXXXXXXXXXXX XXXXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XXXXXXXXXXXXXXX XXXXXXXXXX XXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-P XXXXXXXXXXXXXXX XXXXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-YXX XXX XX XXXXXXXX XXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-YXX XXX XX XXXXXXXX XXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-YXX XXX XX XXXXXXXX XXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-PYXX XXX XX XXXXXXXX XXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-PXX XXX XX XXXXXXXX XXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-YXX XXX XX XXXXXXXX XXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-PYXX XXX XX XXXXXXXX XXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-PXX XXX XX XXXXXXXX XXXXXX XXXXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-PYXX XXX XX XXXXXXXX XXXXXX XXXXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-PXX XXX XX XXXXXXXX XXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′- XXXX XXX XX XXXXXXXX XXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′- XXXX XXX XX XXXXXXXX XXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′- XXXX XXX XX XXXXXXXX XXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XXXX XXX XX XXXXXXXX XXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′- XXXX XXX XX XXXXXXXX XXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XXXX XXX XX XXXXXXXX XXXXXX XXXXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-P XXXX XXX XX XXXXXXXX XXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′- XXXX XXX XX XXXXXXXX XXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′- XXXX XXX XX XXXXXXXX XXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′- XXXX XXX XX XXXXXXXX XXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XXXX XXX XX XXXXXXXX XXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′- XXXX XXX XX XXXXXXXX XXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XXXX XXX XX XXXXXXXX XXXXXX XXXXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-P XXXX XXX XX XXXXXXXX XXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-YXX XXX XX XXXXXXXX XXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-YXX XXX XX XXXXXXXX XXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-YXX XXX XX XXXXXXXX XXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-PYXX XXX XX XXXXXXXX XXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-PXX XXX XX XXXXXXXX XXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-YXX XXX XX XXXXXXXX XXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-PYXX XXX XX XXXXXXXX XXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-PXX XXX XX XXXXXXXX XXXXXX XXXXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-PYXX XXX XX XXXXXXXX XXXXXX XXXXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-PXX XXX XX XXXXXXXX XXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′- XXXX XXX XX XXXXXXXX XXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′- XXXX XXX XX XXXXXXXX XXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′- XXXX XXX XX XXXXXXXX XXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XXXX XXX XX XXXXXXXX XXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′- XXXX XXX XX XXXXXXXX XXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XXXX XXX XX XXXXXXXX XXXXXX XXXXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-P XXXX XXX XX XXXXXXXX XXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′- XXXX XXX XX XXXXXXXX XXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′- XXXX XXX XX XXXXXXXX XXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′- XXXX XXX XX XXXXXXXX XXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XXXX XXX XX XXXXXXXX XXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′- XXXX XXX XX XXXXXXXX XXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XXXX XXX XX XXXXXXXX XXXXXX XXXXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-P XXXX XXX XX XXXXXXXX XXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-Y XX XX XX XX XX XX XX XXXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-Y XX XX XX XX XX XX XX XXXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-Y XX XX XX XX XX XX XX XXXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-PY XX XX XX XX XX XX XX XXXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XX XX XX XX XX XX XX XXXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-Y XX XX XX XX XX XX XX XXXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-PY XX XX XX XX XX XX XX XXXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XX XX XX XX XX XX XX XXXXXXX XXXXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-PY XX XX XX XX XX XX XX XXXXXXX XXXXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-P XX XX XX XX XX XX XX XXXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′- XXXX XX XX XX XX XX XX XXXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′- XXXX XX XX XX XX XX XX XXXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′- XXXX XX XX XX XX XX XX XXXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XXXX XX XX XX XX XX XX XXXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′- XXXX XX XX XX XX XX XX XXXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XXXX XX XX XX XX XX XX XXXXXXX XXXXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-P XXXX XX XX XX XX XX XX XXXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′- XX XX XX XX XX XX XX XX XXXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′- XX XX XX XX XX XX XX XX XXXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′- XX XX XX XX XX XX XX XX XXXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XX XX XX XX XX XX XX XX XXXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′- XX XX XX XX XX XX XX XX XXXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XX XX XX XX XX XX XX XX XXXXXXX XXXXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-P XX XX XX XX XX XX XX XX XXXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-Y XX XX XX XX XX XX XX XXXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-Y XX XX XX XX XX XX XX XXXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-Y XX XX XX XX XX XX XX XXXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-PY XX XX XX XX XX XX XX XXXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XX XX XX XX XX XX XX XXXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-Y XX XX XX XX XX XX XX XXXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-PY XX XX XX XX XX XX XX XXXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XX XX XX XX XX XX XX XXXXXXX XXXXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-PY XX XX XX XX XX XX XX XXXXXXX XXXXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-P XX XX XX XX XX XX XX XXXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′- XXXX XX XX XX XX XX XX XXXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′- XXXX XX XX XX XX XX XX XXXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′- XXXX XX XX XX XX XX XX XXXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XXXX XX XX XX XX XX XX XXXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′- XXXX XX XX XX XX XX XX XXXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XXXX XX XX XX XX XX XX XXXXXXX XXXXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-P XXXX XX XX XX XX XX XX XXXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′- XX XX XX XX XX XX XX XX XXXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′- XX XX XX XX XX XX XX XX XXXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′- XX XX XX XX XX XX XX XX XXXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XX XX XX XX XX XX XX XX XXXXXXX XXXX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′- XX XX XX XX XX XX XX XX XXXXXXX XXXX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XX XX XX XX XX XX XX XX XXXXXXX XXXXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-P XX XX XX XX XX XX XX XX XXXXXXX XXXXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-YXX XXX XXXXXXXX XXXXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-YXX XXX XXXXXXXX XXXXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-YXX XXX XXXXXXXX XXXXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-PYXX XXX XXXXXXXX XXXXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-PXX XXX XXXXXXXX XXXXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-YXX XXX XXXXXXXX XXXXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-PYXX XXX XXXXXXXX XXXXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-PXX XXX XXXXXXXX XXXXXXXXXX XXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-PYXX XXX XXXXXXXX XXXXXXXXXX XXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-PXX XXX XXXXXXXX XXXXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′- XXXX XXX XXXXXXXX XXXXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′- XXXX XXX XXXXXXXX XXXXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′- XXXX XXX XXXXXXXX XXXXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XXXX XXX XXXXXXXX XXXXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′- XXXX XXX XXXXXXXX XXXXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XXXX XXX XXXXXXXX XXXXXXXXXX XXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-P XXXX XXX XXXXXXXX XXXXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′- XXXX XXX XXXXXXXX XXXXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′- XXXX XXX XXXXXXXX XXXXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′- XXXX XXX XXXXXXXX XXXXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XXXX XXX XXXXXXXX XXXXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′- XXXX XXX XXXXXXXX XXXXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XXXX XXX XXXXXXXX XXXXXXXXXX XXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-P XXXX XXX XXXXXXXX XXXXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-YXX XXX XXXXXXXX XXXXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-YXX XXX XXXXXXXX XXXXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-YXX XXX XXXXXXXX XXXXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-PYXX XXX XXXXXXXX XXXXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-PXX XXX XXXXXXXX XXXXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-YXX XXX XXXXXXXX XXXXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-PYXX XXX XXXXXXXX XXXXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-PXX XXX XXXXXXXX XXXXXXXXXX XXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-PYXX XXX XXXXXXXX XXXXXXXXXX XXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-PXX XXX XXXXXXXX XXXXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′- XXXX XXX XXXXXXXX XXXXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′- XXXX XXX XXXXXXXX XXXXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′- XXXX XXX XXXXXXXX XXXXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XXXX XXX XXXXXXXX XXXXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′- XXXX XXX XXXXXXXX XXXXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XXXX XXX XXXXXXXX XXXXXXXXXX XXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-P XXXX XXX XXXXXXXX XXXXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′- XXXX XXX XXXXXXXX XXXXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′- XXXX XXX XXXXXXXX XXXXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′- XXXX XXX XXXXXXXX XXXXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XXXX XXX XXXXXXXX XXXXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′- XXXX XXX XXXXXXXX XXXXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XXXX XXX XXXXXXXX XXXXXXXXXX XXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-P XXXX XXX XXXXXXXX XXXXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-Y XX XX XX XX XX XX XX XXXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-Y XX XX XX XX XX XX XX XXXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-Y XX XX XX XX XX XX XX XXXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-PY XX XX XX XX XX XX XX XXXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XX XX XX XX XX XX XX XXXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-Y XX XX XX XX XX XX XX XXXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-PY XX XX XX XX XX XX XX XXXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XX XX XX XX XX XX XX XXXXXXXXX XXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-PY XX XX XX XX XX XX XX XXXXXXXXX XXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-P XX XX XX XX XX XX XX XXXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′- XXXX XX XX XX XX XX XX XXXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′- XXXX XX XX XX XX XX XX XXXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′- XXXX XX XX XX XX XX XX XXXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XXXX XX XX XX XX XX XX XXXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′- XXXX XX XX XX XX XX XX XXXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XXXX XX XX XX XX XX XX XXXXXXXXX XXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-P XXXX XX XX XX XX XX XX XXXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′- XX XX XX XX XX XX XX XX XXXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′- XX XX XX XX XX XX XX XX XXXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′- XX XX XX XX XX XX XX XX XXXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XX XX XX XX XX XX XX XX XXXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′- XX XX XX XX XX XX XX XX XXXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XX XX XX XX XX XX XX XX XXXXXXXXX XXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-P XX XX XX XX XX XX XX XX XXXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-Y XX XX XX XX XX XX XX XXXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-Y XX XX XX XX XX XX XX XXXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-Y XX XX XX XX XX XX XX XXXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-PY XX XX XX XX XX XX XX XXXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XX XX XX XX XX XX XX XXXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-Y XX XX XX XX XX XX XX XXXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-PY XX XX XX XX XX XX XX XXXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XX XX XX XX XX XX XX XXXXXXXXX XXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-PY XX XX XX XX XX XX XX XXXXXXXXX XXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-P XX XX XX XX XX XX XX XXXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′- XXXX XX XX XX XX XX XX XXXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′- XXXX XX XX XX XX XX XX XXXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′- XXXX XX XX XX XX XX XX XXXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XXXX XX XX XX XX XX XX XXXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′- XXXX XX XX XX XX XX XX XXXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XXXX XX XX XX XX XX XX XXXXXXXXX XXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-P XXXX XX XX XX XX XX XX XXXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′- XX XX XX XX XX XX XX XX XXXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′- XX XX XX XX XX XX XX XX XXXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′- XX XX XX XX XX XX XX XX XXXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XX XX XX XX XX XX XX XX XXXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′- XX XX XX XX XX XX XX XX XXXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XX XX XX XX XX XX XX XX XXXXXXXXX XXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-P XX XX XX XX XX XX XX XX XXXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-Y XX XX XX XX XX XX XX XXXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-Y XX XX XX XX XX XX XX XXXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-Y XX XX XX XX XX XX XX XXXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-PY XX XX XX XX XX XX XX XXXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XX XX XX XX XX XX XX XXXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-Y XX XX XX XX XX XX XX XXXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-PY XX XX XX XX XX XX XX XXXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XX XX XX XX XX XX XX XXXXXXXXX XXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-PY XX XX XX XX XX XX XX XXXXXXXXX XXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-P XX XX XX XX XX XX XX XXXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′- XXXX XX XX XX XX XX XX XXXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′- XXXX XX XX XX XX XX XX XXXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′- XXXX XX XX XX XX XX XX XXXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XXXX XX XX XX XX XX XX XXXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′- XXXX XX XX XX XX XX XX XXXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XXXX XX XX XX XX XX XX XXXXXXXXX XXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-P XXXX XX XX XX XX XX XX XXXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′- XX XX XX XX XX XX XX XX XXXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′- XX XX XX XX XX XX XX XX XXXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′- XX XX XX XX XX XX XX XX XXXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XX XX XX XX XX XX XX XX XXXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′- XX XX XX XX XX XX XX XX XXXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXDD-3′
3′-P XX XX XX XX XX XX XX XX XXXXXXXXX XXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXDDP-3′
3′-P XX XX XX XX XX XX XX XX XXXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-Y XX XX XX XX XX XX XX XXXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-Y XX XX XX XX XX XX XX XXXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-Y XX XX XX XX XX XX XX XXXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-PY XX XX XX XX XX XX XX XXXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XX XX XX XX XX XX XX XXXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-Y XX XX XX XX XX XX XX XXXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-PY XX XX XX XX XX XX XX XXXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XX XX XX XX XX XX XX XXXXXXXXX XXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-PY XX XX XX XX XX XX XX XXXXXXXXX XXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-P XX XX XX XX XX XX XX XXXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′- XXXX XX XX XX XX XX XX XXXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′- XXXX XX XX XX XX XX XX XXXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′- XXXX XX XX XX XX XX XX XXXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XXXX XX XX XX XX XX XX XXXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′- XXXX XX XX XX XX XX XX XXXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XXXX XX XX XX XX XX XX XXXXXXXXX XXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-P XXXX XX XX XX XX XX XX XXXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′- XX XX XX XX XX XX XX XX XXXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′- XX XX XX XX XX XX XX XX XXXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′- XX XX XX XX XX XX XX XX XXXXXXXXX XXP-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XX XX XX XX XX XX XX XX XXXXXXXXX XX-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′- XX XX XX XX XX XX XX XX XXXXXXXXX XX-5′
Or
5′-XXXXXXXXXXXXXXXXXXXXXXXXX-3′
3′-P XX XX XX XX XX XX XX XX XXXXXXXXX XXP-5′
Or
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXP-3′
3′-P XX XX XX XX XX XX XX XX XXXXXXXXX XXP-5′
Wherein, " X "=RNA, " X"=2 '-the O-methyl RNA, the outstanding domain that " Y " is made up of 1 to 4 RNA monomer, said RNA monomer randomly be 2 '-O-methyl RNA monomer, the residue of leukorrhagia line is 2 '-O-methyl RNA monomer, " D "=DNA and " P "=peptide.Cochain is a sense strand, and chain is an antisense strand down.
In another embodiment, dsRNA comprises the chain with equal length.
In this type of embodiment, dsRNA comprises:
5′-XXXXXXXXXXXXXXXXXXXXXXXXXMMP-3′
3′-XXXXXXXXXXXXXXXXXXXXXXXXXMM-5’
Wherein, " X "=RNA, " M " are nucleic acid residue (RNA, DNA or nucleic acid non-natural or through modifying) and " P "=peptide.Any this type of residue of this type of reagent can randomly be 2 '-3 of (second) chain ' end residue begins from the bottom 2 for O-methyl RNA monomer-shown in above unsymmetrical reagent '-the monomeric positioned alternate of O-methyl RNA also can be used for above-mentioned flush end-flush end dsRNA reagent.
In this type of embodiment, dsRNA comprises:
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXMM-3′
3′-XXXXXXXXXXXXXXXXXXXXXXXXXMM-5′
Wherein, " X "=RNA, " M " are nucleic acid residue (RNA, DNA or nucleic acid non-natural or through modifying) and " P "=peptide.Any this type of residue of this type of reagent can randomly be 2 '-3 of (second) chain ' end residue begins from the bottom 2 for O-methyl RNA monomer-shown in above unsymmetrical reagent '-the monomeric positioned alternate of O-methyl RNA also can be used for above-mentioned flush end-flush end dsRNA reagent.
In this type of embodiment, dsRNA comprises:
5′-XXXXXXXXXXXXXXXXXXXXXXXXXMM-3′
3′-XXXXXXXXXXXXXXXXXXXXXXXXXMMP-5’
Wherein, " X "=RNA, " M " are nucleic acid residue (RNA, DNA or nucleic acid non-natural or through modifying) and " P "=peptide.Any this type of residue of this type of reagent can randomly be 2 '-3 of (second) chain ' end residue begins from the bottom 2 for O-methyl RNA monomer-shown in above unsymmetrical reagent '-the monomeric positioned alternate of O-methyl RNA also can be used for above-mentioned flush end-flush end dsRNA reagent.
In this type of embodiment, dsRNA comprises:
5′-XXXXXXXXXXXXXXXXXXXXXXXXXMM-3′
3′-PXXXXXXXXXXXXXXXXXXXXXXXXXMM-5’
Wherein, " X "=RNA, " M " are nucleic acid residue (RNA, DNA or nucleic acid non-natural or through modifying) and " P "=peptide.Any this type of residue of this type of reagent can randomly be 2 '-3 of (second) chain ' end residue begins from the bottom 2 for O-methyl RNA monomer-shown in above unsymmetrical reagent '-the monomeric positioned alternate of O-methyl RNA also can be used for above-mentioned flush end-flush end dsRNA reagent.
In this type of embodiment, dsRNA comprises:
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXMMP-3′
3′-XXXXXXXXXXXXXXXXXXXXXXXXXMM-5’
" X "=RNA wherein, " M " are nucleic acid residue (RNA, DNA or nucleic acid non-natural or through modifying) and " P "=peptide.Any this type of residue of this type of reagent can randomly be 2 '-3 of (second) chain ' end residue begins from the bottom 2 for O-methyl RNA monomer-shown in above unsymmetrical reagent '-the monomeric positioned alternate of O-methyl RNA also can be used for above-mentioned flush end-flush end dsRNA reagent.
In this type of embodiment, dsRNA comprises:
5′-XXXXXXXXXXXXXXXXXXXXXXXXXMM-3′
3′-PXXXXXXXXXXXXXXXXXXXXXXXXXMMP-5’
" X "=RNA wherein, " M " are nucleic acid residue (RNA, DNA or nucleic acid non-natural or through modifying) and " P "=peptide.Any this type of residue of this type of reagent can randomly be 2 '-3 of (second) chain ' end residue begins from the bottom 2 for O-methyl RNA monomer-shown in above unsymmetrical reagent '-the monomeric positioned alternate of O-methyl RNA also can be used for above-mentioned flush end-flush end dsRNA reagent.
In this type of embodiment, dsRNA comprises:
5′-PXXXXXXXXXXXXXXXXXXXXXXXXXMMP-3′
3′-PXXXXXXXXXXXXXXXXXXXXXXXXXMMP-5’
" X "=RNA wherein, " M " are nucleic acid residue (RNA, DNA or nucleic acid non-natural or through modifying) and " P "=peptide.Any this type of residue of this type of reagent can randomly be 2 '-3 of (second) chain ' end residue begins from the bottom 2 for O-methyl RNA monomer-shown in above unsymmetrical reagent '-the monomeric positioned alternate of O-methyl RNA also can be used for above-mentioned flush end-flush end dsRNA reagent.
The exemplary configurations of any the following stated is also contained in the present invention; At least one end of at least one chain at least one peptide wherein of the present invention and first chain or second chain is puted together, and perhaps puts together with first chain of dsRNA of the present invention or at least one the chain inside in second chain.
In another embodiment; DsiRNA comprises and has equal length and have 1 to 3 as the chain of the cracked mispairing residue of directed Dicer (specifically; When first chain and second chain are annealed each other, the corresponding residue mispairing of 5 ' petiolarea on one or more and second chain the position 1,2 or 3 on first chain of DsiRNA (when the time) from 3 ' end residue numbering).Exemplary 27 mer DsiRNA reagent with two terminal mispairing residues are shown as:
Figure BDA0000133674250002081
" X "=RNA wherein, " M "=when chain is annealed not with the nucleic acid residue (RNA, DNA or nucleic acid non-natural or) of otherwise corresponding " M " the residue base pairing (hydrogen bond) of complementary chain through modifying.Any this type of residue of this type of reagent can randomly be 2 '-O-methyl RNA monomer just like 3 ' of (second) chain end residue begins 2 from the bottom shown in the above unsymmetrical reagent '-the monomeric positioned alternate of O-methyl RNA also can be used for above-mentioned " flush end/frayed end (fray) " dsRNA reagent.Cochain (first chain) is a sense strand, and chain (second chain) is an antisense strand down.
In some other embodiment; The present invention provides RNA to disturb (RNAi) compositions; Said RNA disturbs (RNAi) compositions in the district of double-strandednucleic acid (dsNA), to have the deoxyribonucleotide of one or more base pairings, and said double-strandednucleic acid is positioned at 3 ' end and the corresponding 5 ' end of the antisense strand Dicer cracking site estimated of the sense strand Dicer cracking site of expectation.It is the dsNA of precursor molecule that compositions of the present invention comprises, that is, dsNA of the present invention processes in body to generate active small RNA (siRNA).DsNA is processed into the active siRNA that incorporates among the RISC through Dicer.
In certain embodiments, DsiRNA reagent of the present invention can have any following exemplary configurations:
In this type of embodiment, DsiRNA comprises:
5′-XXXXXXXXXXXXXXXXXXXXXXXX N＊D NDD-3′
3′-YXXXXXXXXXXXXXXXXXXXXXXXX N＊D NXX-5’
Wherein, " X "=RNA, the optional outstanding domain that " Y " is made up of 0 to 10 RNA monomer; Said RNA monomer randomly is 2 '-O-methyl RNA monomer, in certain embodiments, the outstanding domain that " Y " is made up of 1 to 4 RNA monomer; Said RNA monomer randomly is 2 '-O-methyl RNA monomer; " D "=DNA, and " N "=1 randomly is 1 to 8 still to 50 or more." N* "=0 randomly is 0,1,2,3,4,5 or 6 still to 15 or more.In one embodiment, cochain is a sense strand, and chain is an antisense strand down.Perhaps, following chain is a sense strand, and cochain is an antisense strand.
In related embodiment, DsiRNA comprises:
5′-XXXXXXXXXXXXXXXXXXXXXXXX N＊D NDD-3′
3′-YXXXXXXXXXXXXXXXXXXXXXXXX N＊D NDD-5’
Wherein, " X "=RNA, the optional outstanding domain that " Y " is made up of 0 to 10 RNA monomer; Said RNA monomer randomly is 2 '-O-methyl RNA monomer, in certain embodiments, the outstanding domain that " Y " is made up of 1 to 4 RNA monomer; Said RNA monomer randomly is 2 '-O-methyl RNA monomer; " D "=DNA, and " N "=1 randomly is 1 to 8 still to 50 or more." N* "=0 randomly is 0,1,2,3,4,5 or 6 still to 15 or more.In one embodiment, cochain is a sense strand, and chain is an antisense strand down.Perhaps, following chain is a sense strand, and cochain is an antisense strand.
In another this type of embodiment, DsiRNA comprises:
5′-XXXXXXXXXXXXXXXXXXXXXXXX N＊D NDD-3′
3′-Y XX XX XX XX XX XX XX XX XXXXXXXX N＊D NZZ-5’
Wherein, " X "=RNA, " X"=2 '-the O-methyl RNA, the optional outstanding domain that " Y " is made up of 0 to 10 RNA monomer, said RNA monomer randomly be 2 '-O-methyl RNA monomer; in certain embodiments, the outstanding domain that " Y " is made up of 1 to 4 RNA monomer, said RNA monomer randomly be 2 '-O-methyl RNA monomer; " D "=DNA; " Z "=DNA or RNA, and " N "=1 randomly is 1 to 8 still to 50 or more." N* "=0 randomly is 0,1,2,3,4,5 or 6 still to 15 or more.In one embodiment, cochain is a sense strand, and chain is an antisense strand down.Perhaps, following chain is a sense strand, and cochain is antisense strand, wherein 2 '-O-methyl RNA monomer is positioned alternative residue place along cochain (rather than in the above-mentioned sketch map the present following chain that illustrates).
In another this type of embodiment, DsiRNA comprises:
5′-XXXXXXXXXXXXXXXXXXXXXXXX N＊D NDD-3′
3′-Y XX XX XX XX XX XX XX XX XX XXXXXX N＊D NZZ-5’
Wherein, " X "=RNA, " X"=2 '-the O-methyl RNA, the optional outstanding domain that " Y " is made up of 0 to 10 RNA monomer, said RNA monomer randomly be 2 '-O-methyl RNA monomer; in certain embodiments, the outstanding domain that " Y " is made up of 1 to 4 RNA monomer, said RNA monomer randomly be 2 '-O-methyl RNA monomer; " D "=DNA; " Z "=DNA or RNA, and " N "=1 randomly is 1 to 8 still to 50 or more." N* "=0 randomly is 0,1,2,3,4,5 or 6 still to 15 or more.In one embodiment, cochain is a sense strand, and chain is an antisense strand down.Perhaps, following chain is a sense strand, and cochain is antisense strand, wherein 2 '-O-methyl RNA monomer is positioned alternative residue place along cochain (rather than in the above-mentioned sketch map the present following chain that illustrates).
In another embodiment, DsiRNA comprises:
5′-XXXXXXXXXXXXXXXXXXXXXXXX N＊[X1/D1] NDD-3′
3′-YXXXXXXXXXXXXXXXXXXXXXXXX N＊[X2/D2] NZZ-5’
Wherein, " X "=RNA, the optional outstanding domain that " Y " is made up of 0 to 10 RNA monomer; Said RNA monomer randomly is 2 '-O-methyl RNA monomer, in certain embodiments, the outstanding domain that " Y " is made up of 1 to 4 RNA monomer; Said RNA monomer randomly is 2 '-O-methyl RNA monomer; " D "=DNA, " Z "=DNA or RNA, and " N "=1 is to 50 or more; But randomly be 1 to 8, wherein at least one D1N be present in the cochain and with following chain in corresponding D2N base pairing.Randomly, D1 NAnd D1 N+1With corresponding D2 NAnd D2 N+1Base pairing; D1 N, D1 N+1And D1 N+2With corresponding D2 N, D1 N+1And D1 N+2Base pairing etc." N* "=0 randomly is 0,1,2,3,4,5 or 6 still to 15 or more.In one embodiment, cochain is a sense strand, and chain is an antisense strand down.Perhaps, following chain is a sense strand, and cochain is antisense strand, wherein 2 '-O-methyl RNA monomer is positioned alternative residue place along cochain (rather than in the above-mentioned sketch map the present following chain that illustrates).
In any structure in the above-mentioned structure that illustrates, 5 of sense strand or antisense strand ' end randomly comprises phosphate group.
In another embodiment; DNA:DNA-prolongs DsiRNA and comprises and have equal length and have 1 to 3 as the cracked mispairing residue of directed Dicer (specifically; When first chain and second chain are annealed each other, the corresponding residue mispairing of 5 ' petiolarea on the one or more positions the position 1,2,3 on first chain of DsiRNA (when the time) and second chain) chain from 3 ' end residue numbering.Exemplary DNA:DNA-with two terminal mispairing residues prolongs DsiRNA reagent and is shown as:
Figure BDA0000133674250002111
Wherein, " X "=RNA; " M "=when chain is annealed not with the nucleic acid residue (RNA, DNA or nucleic acid non-natural or) of corresponding " M " the residue base pairing (hydrogen bond) of other complementary strand through modifying, " D "=DNA and " N "=1 is to 50 or more, but randomly is 1 to 8." N* "=0 randomly is 0,1,2,3,4,5 or 6 still to 15 or more.Any this type of residue of this type of reagent can randomly be 2 '-the monomeric positioned alternate of 2 '-O-methyl RNA that O-methyl RNA monomer-3 of (second) chain ' end residue begins from the bottom shown in above unsymmetrical reagent also can be used for above-mentioned " flush end/frayed end " dsRNA reagent.In one embodiment, cochain (first chain) is a sense strand, and chain (second chain) is an antisense strand down.Perhaps, following chain is a sense strand, and cochain is an antisense strand.The modification and the DNA:DNA extension mode that are parallel to those patterns of the asymmetric/outstanding reagent shown in above also can be incorporated in this type of " flush end/frayed end " reagent.
In one embodiment; Suppose that length extension DsiRNA reagent comprises deoxyribonucleotide; Said deoxyribonucleotide is positioned to be modelled as the acting site through the cracked certain orientation of Dicer, but need there be the base pairing deoxyribonucleotide in said deoxyribonucleotide in the dsNA structure.The exemplary configurations of this molecule is shown as:
5′-XXXXXXXXXXXXXXXXXXXDDXX-3′
3′-YXXXXXXXXXXXXXXXXXDDXXXX-5′
Wherein, " X "=RNA, the optional outstanding domain that " Y " is made up of 0 to 10 RNA monomer, said RNA monomer randomly be 2 '-O-methyl RNA monomer; In certain embodiments; The outstanding domain that " Y " is made up of 1 to 4 RNA monomer, said RNA monomer randomly be 2 '-O-methyl RNA monomer, and " D "=DNA.In one embodiment, cochain is a sense strand, and chain is an antisense strand down.Perhaps, following chain is a sense strand, and cochain is an antisense strand.Said structure forces Dicer to be cracked into minimum 21mer duplex through being modelled as, as the principal mode after the Dicer processing.The following chain of said structure is in the embodiment of antisense strand therein, last residue and the penult residue place that two deoxyribonucleotide residues are positioned antisense strand 5 ' end possibly reduce to miss the target effect (as existing research show at least from 2 of the penult position of antisense strand 5 ' end '-modification of O-methyl reduces the effect of missing the target; For example, referring to US2007/0223427).
In one embodiment, DsiRNA comprises:
5′-D NXXXXXXXXXXXXXXXXXXXXXXXX N＊Y-3′
3′-D NXXXXXXXXXXXXXXXXXXXXXXXX N＊-5’
Wherein, " X "=RNA, the optional outstanding domain that " Y " is made up of 0 to 10 RNA monomer; Said RNA monomer randomly is 2 '-O-methyl RNA monomer, in certain embodiments, the outstanding domain that " Y " is made up of 1 to 4 RNA monomer; Said RNA monomer randomly is 2 '-O-methyl RNA monomer; " D "=DNA, and " N "=1 randomly is 1 to 8 still to 50 or more." N* "=0 randomly is 0,1,2,3,4,5 or 6 still to 15 or more.In one embodiment, cochain is a sense strand, and chain is an antisense strand down.Perhaps, following chain is a sense strand, and cochain is an antisense strand.
In related embodiment, DsiRNA comprises:
5′-D NXXXXXXXXXXXXXXXXXXXXXXXX N＊DD-3′
3′-D NXXXXXXXXXXXXXXXXXXXXXXXX N＊XX-5’
Wherein, " X "=RNA, randomly be 2 '-O-methyl RNA monomer, " D "=DNA, " N "=1 is to 50 or more, but randomly is 1 to 8." N* "=0 randomly is 0,1,2,3,4,5 or 6 still to 15 or more.In one embodiment, cochain is a sense strand, and chain is an antisense strand down.Perhaps, following chain is a sense strand, and cochain is an antisense strand.
In another this type of embodiment, DsiRNA comprises:
5′-D NXXXXXXXXXXXXXXXXXXXXXXXX N＊DD-3′
3′-D N XX XX XX XX XX XX XX XX XXXXXXXX N＊Z?Z-5’
Wherein, " X "=RNA, randomly be 2 '-O-methyl RNA monomer, " D "=DNA, " N "=1 is to 50 or more, but randomly is 1 to 8." N* "=0 randomly is 0,1,2,3,4,5 or 6 still to 15 or more." Z "=DNA or RNA.In one embodiment, cochain is a sense strand, and chain is an antisense strand down.Perhaps, following chain is a sense strand, and cochain is antisense strand, and 2 '-O-methyl RNA monomer is positioned alternative residue place along cochain (rather than in the above-mentioned sketch map the present following chain that illustrates).
In another this type of embodiment, DsiRNA comprises:
5′-D NZZXXXXXXXXXXXXXXXXXXXXXXXX N＊DD-3′
3′-D N XXXX XX XX XX XX XX XX XX XX XXXXXX N＊ZZ-5’
Wherein, " X "=RNA, " X"=2 '-the O-methyl RNA, " D "=DNA, " Z "=DNA or RNA, and " N "=1 randomly is 1 to 8 still to 50 or more." N* "=0 randomly is 0,1,2,3,4,5 or 6 still to 15 or more.In one embodiment, cochain is a sense strand, and chain is an antisense strand down.Perhaps, following chain is a sense strand, and cochain is antisense strand, wherein 2 '-O-methyl RNA monomer is positioned alternative residue place along cochain (rather than in the above-mentioned sketch map the present following chain that illustrates).
In another this type of embodiment, DsiRNA comprises:
5′-D NZZXXXXXXXXXXXXXXXXXXXXXXXX N＊Y-3′
3′-D N XXXX XX XX XX XX XX XX XX XX XXXXXX N＊-5’
Wherein, " X "=RNA, " X"=2 '-the O-methyl RNA, " D "=DNA, " Z "=DNA or RNA, or " N "=1 randomly is 1 to 8 still to 50 or more." N* "=0 randomly is 0,1,2,3,4,5 or 6 still to 15 or more.The optional outstanding domain that " Y " is made up of 0 to 10 RNA monomer; Said RNA monomer randomly is 2 '-O-methyl RNA monomer; In certain embodiments, the outstanding domain that " Y " is made up of 1 to 4 RNA monomer, said RNA monomer randomly be 2 '-O-methyl RNA monomer.In one embodiment, cochain is a sense strand, and chain is an antisense strand down.Perhaps, following chain is a sense strand, and cochain is antisense strand, wherein 2 '-O-methyl RNA monomer is positioned alternative residue place along cochain (rather than in the above-mentioned sketch map the present following chain that illustrates).
In another embodiment, DsiRNA comprises:
5′-[X1/D1] NXXXXXXXXXXXXXXXXXXXXXXXX N＊DD-3′
3′-[X2/D2] NXXXXXXXXXXXXXXXXXXXXXXXX N＊ZZ-5’
Wherein, " X "=RNA, " D "=DNA, " Z "=DNA or RNA, and " N "=1 is to 50 or more, but randomly be 1 to 8, wherein at least one D1N be present in the cochain and with following chain in corresponding D2N base pairing.Randomly, D1 NAnd D1 N+1With corresponding D2 NAnd D2 N+1Base pairing; D1 N, D1 N+1And D1 N+2With corresponding D2 N, D1 N+1And D1 N+2Base pairing etc." N* "=0 randomly is 0,1,2,3,4,5 or 6 still to 15 or more.In one embodiment, cochain is a sense strand, and chain is an antisense strand down.Perhaps, following chain is a sense strand, and cochain is antisense strand, and wherein 2 '-O-methyl RNA monomer is positioned alternative residue place along cochain (rather than in the above-mentioned sketch map the present following chain that illustrates).
In related embodiment, DsiRNA comprises:
5′-[X1/D1] NXXXXXXXXXXXXXXXXXXXXXXXX N＊Y-3′
3′-[X?2/D?2] NXXXXXXXXXXXXXXXXXXXXXXXX N＊-5’
Wherein, " X "=RNA, " D "=DNA; The optional outstanding domain that " Y " is made up of 0 to 10 RNA monomer, said RNA monomer randomly be 2 '-O-methyl RNA monomer, in certain embodiments; The outstanding domain that " Y " is made up of 1 to 4 RNA monomer, said RNA monomer randomly be 2 '-O-methyl RNA monomer, and " N "=1 is to 50 or more; But randomly be 1 to 8, wherein at least one D1N be present in the cochain and with following chain in corresponding D2N base pairing.Randomly, D1 NAnd D1 N+1With corresponding D2 NAnd D2 N+1Base pairing; D1 N, D1 N+1And D1 N+2With corresponding D2 N, D1 N+1And D1 N+2Base pairing etc." N* "=0 randomly is 0,1,2,3,4,5 or 6 still to 15 or more.In one embodiment, cochain is a sense strand, and chain is an antisense strand down.Perhaps, following chain is a sense strand, and cochain is antisense strand, and 2 '-O-methyl RNA monomer is positioned alternative residue place along cochain (rather than in the above-mentioned sketch map the present following chain that illustrates).
In any above-mentioned structure that illustrates, 5 of sense strand or antisense strand ' end randomly comprises phosphate.
In another embodiment; The DsiRNA that DNA:DNA-extends comprises and has equal length and have 1 to 3 as the cracked mispairing residue of directed Dicer (specifically; When first chain and second chain are annealed each other, the corresponding residue mispairing of 5 ' petiolarea on one or more and second chain the position 1,2,3 on first chain of DsiRNA (when the time)) chain from 3 ' end residue numbering.Exemplary DNA:DNA-with two terminal mispairing residues extends DsiRNA reagent and is shown as:
Figure BDA0000133674250002141
Figure BDA0000133674250002151
Wherein, " X "=RNA; " M "=when chain is annealed not otherwise with the nucleic acid residue (RNA, DNA or nucleic acid non-natural or) of corresponding " M " the residue base pairing (hydrogen bond) of other complementary strand through modifying, " D "=DNA and " N "=1 is to 50 or more, but randomly is 1 to 8." N* "=0 randomly is 0,1,2,3,4,5 or 6 still to 15 or more.Any this type of residue of this type of reagent can randomly be 2 '-3 of (second) chain ' end residue begins from the bottom 2 for O-methyl RNA monomer-shown in above unsymmetrical reagent '-the monomeric positioned alternate of O-methyl RNA also can be used for above-mentioned " flush end/frayed end " dsRNA reagent.In one embodiment, cochain (first chain) is a sense strand, and chain (second chain) is an antisense strand down.Perhaps, following chain is a sense strand, and cochain is an antisense strand.Those modification and the DNA:DNA extension mode that is parallel to the asymmetric/outstanding agent shown in above also can be incorporated in this type of " flush end/frayed end " reagent.
In another embodiment; Suppose that the DsiRNA reagent that length is extended comprises deoxyribonucleotide; Said deoxyribonucleotide is positioned to be modelled as the acting site through the cracked certain orientation of Dicer, but need there be the deoxyribonucleotide of base pairing in said deoxyribonucleotide in the dsNA structure.The exemplary configurations of this molecule is shown as:
5′-XXDDXXXXXXXXXXXXXXXXXXXX N＊Y-3′
3′-DDXXXXXXXXXXXXXXXXXXXXXX N＊-5′
Wherein, " X "=RNA, the optional outstanding domain that " Y " is made up of 0 to 10 RNA monomer, said RNA monomer randomly be 2 '-O-methyl RNA monomer; In certain embodiments; The outstanding domain that " Y " is made up of 1 to 4 RNA monomer, said RNA monomer randomly be 2 '-O-methyl RNA monomer, and " D "=DNA." N* "=0 randomly is 0,1,2,3,4,5 or 6 still to 15 or more.In one embodiment, cochain is a sense strand, and chain is an antisense strand down.Perhaps, following chain is a sense strand, and cochain is an antisense strand.Said structure makes Dicer be cracked into the principal mode after minimum 21mer duplex is processed as Dicer through being modelled as.The following chain of said structure is in the embodiment of antisense strand therein, last that two deoxyribonucleotide residues are positioned antisense strand 5 ' end and penult residue place possibly reduce to miss the target effect (show like existing research: from 2 of the position of penult at least of antisense strand 5 ' end '-modification of O-methyl reduces the effect of missing the target; For example, referring to US 2007/0223427).
In certain embodiments, " D " residue in any said structure comprises at least one PS-DNA or PS-RNA.Randomly, " D " residue in any said structure comprises that at least one suppresses the cracked nucleotide through modifying of Dicer.
Though above-mentioned " DNA extends " DsiRNA reagent can be categorized as " left side extension " or " right side extension "; But also can be by this paper about " extend on the right side " and " extend in the left side " the said similar mode of reagent; Produce and use and in single reagent, comprise the DsiRNA reagent that contains DNA sequence that extend in the left side and extend on the right side (being that dsDNA extends in the both sides of core dsRNA structure periphery for example) simultaneously.
In some embodiments, DsiRNA of the present invention further comprises the sense strand of the DsiRNA reagent that connects the DNA:DNA-extension and the coupling part or the domain of antisense strand.Randomly, 3 ' end of this coupling part domain connection sense strand and 5 ' end of antisense strand.The coupling part maybe be for chemistry (non-nucleotide) connexon, such as oligomerization methylene glycol connexon, oligomeric ethylene glycol connexon or other art-recognized connexon part.Perhaps, connexon can be nucleotide linker, randomly comprises extended loop and/or tetranucleotide ring.
In one embodiment; DsiRNA reagent has dissymmetrical structure; And sense strand has 25 base pair length; And antisense strand have length be 27 base pairs and have 3 of 1 to 4 base ' outstanding (for example, 3 of a base ' 3 ' outstanding or four bases of 3 ' outstanding, three bases of outstanding, two bases 3 ' outstanding).In another embodiment, this DsiRNA reagent has dissymmetrical structure, and 2 Deoxydization nucleotides are further contained at its 3 ' end place at sense strand.
In another embodiment; DsiRNA reagent has dissymmetrical structure; And the length of antisense strand is 25 base pairs; And the length of sense strand be 27 base pairs and have 3 of 1 to 4 base ' outstanding (for example, 3 of a base ' 3 ' outstanding or four bases of 3 ' outstanding, three bases of outstanding, two bases 3 ' outstanding).In another embodiment, this DsiRNA reagent has dissymmetrical structure, and 2 Deoxydization nucleotides are further contained at its 3 ' end place at antisense strand.
Concerning above-mentioned flush end of the present invention/frayed end reagent; Think that the accurate sequence of frayed end structure is not most important concerning effect, for example one or two residue in 3 of first chain ' end residue only need be not complementary with corresponding 5 ' end residue of second chain.In certain embodiments, DsiRNA reagent of the present invention needs the corresponding residue complementation of at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25 of (for example) first chain or at least 26 residues and second chain.In some related embodiment, the complementary first chain residue of these corresponding residues with second chain randomly is continuous residue.Additionally and/or alternatively; Some mispairing residue can also be positioned (for example) sense strand half 5 ' in; This means in certain embodiments; Complete complementation between first chain and second chain can not keep in whole first chain and second chain; Even do not comprise the diffusing damage structure at first chain 3 ' end/the second chain 5 ' end place-in this type of embodiment; First chain can be effective first chain, and between first chain and second chain complementary degree of (two the 5 ' end residue that randomly, does not comprise two 3 ' end residue/second chain of first chain that loose to decrease reagent) be residue at least 80%, residue at least 85%, residue at least 90%, residue at least 90%, residue at least 96%.In certain embodiments, second chain of DsiRNA of the present invention with respect to first chain or with respect to the complementary degree of target RNA sequence can for first chain or with the complementary level of the target RNA sequence of first chain that is equal to above-mentioned this DsiRNA.
RNA processing
siRNA
Through being present in the process that long dsRNA molecule in the cell triggers siRNA mediate rna i.During the beginning step of RNAi; Through Dicer (the conservative family of containing the enzyme in two rnase iii spline structure territories); These dsRNA molecule cracking are become siRNA duplex (siRNA) (Bernstein etc., 2001 of 21 to 23 nucleotide (nt); Elbashir etc., 2001).SiRNA is characterised in that on every chain 3 of the double-stranded tagma that has 19 to 21 base pairs and 2 nucleotide ' outstanding.During the effector step of RNAi, siRNA begins to incorporate into the multimeric protein complex that is called the inductive reticent complex of RNA (RISC), and wherein their are with the guides of the complete complementary mRNA substrate that is used to degrade of electing.Through mRNA with the complementary district of siRNA in carry out the cracking of inscribe nucleotide initiate the degraded.Or rather, at the 10 nucleotide places, position that come bootstrap siRNA5 ' end with mRNA cracking (Elbashir etc., 2001Genes&Dev.15:188-200; Nykanen etc., 2001Cell 107:309-321; Martinez etc., 2002Cell 110:563-574).To be responsible for this cracked Cobra venom endonuclease and be identified as Argonaute2 (Ago2; Liu etc., Science, 305:1437-41).
miRNA
Most of people miRNA (70%) (with general other mammiferous most of miRNA) are transcribed by intron and/or exon and form, and about 30% be positioned at intergenic region (Rodriguez etc., Genome Res.2004,14 (10A), 1902-1910).In humans and animals, usually by rna plymerase ii transcribe miRNA (Farh etc., Science 2005; 310 (5755), 1817-1821) and in some cases transcribe miRNA (Borchert etc. by polymerase III; Nat.Struct.Mol.Biol.2006,13 (12), 1097-1101).By rna plymerase iii transcribe some encoding viral miRNA (Pfeffer etc., Nat.Methods 2005,2 (4), 269-276; Andersson etc., J.Virol.2005,79 (15), 9556-9565), and some encoding viral miRNA be positioned at viral gene ORFs (Pfeffer etc., Nat.Methods 2005,2 (4), 269-276; Samols etc., J.Virol.2005,79 (14), 9301-9305).MiRNA transcribes and can cause producing the main transcript of big monocistron, bicistronic mRNA or polycistron (pri-miRNA).The length range of single pri-miRNA possibly and have 5 ' 7-methylguanosine (m7) medicated cap and 3 ' for about 200 nucleotide (nt) to thousands of bases (kb) and gather (A) afterbody.Typically, ripe miRNA sequence is positioned the district of incomplete stem ring sequence in the pri-miRNA (Cullen, Mol.Cell 2004,16 (6), 861-865).
Sophisticated first step of miRNA is through complex identification of rnase iii Drosha-DGCR8 nuclear microprocessor and cracking pri-miRNA in the nucleus; Thereby discharge the precursor molecule be called the containing of precursor miRNA～70nt hair clip; Wherein have single phosphoric acid and have the 2-nt that has hydroxyl and give prominence to (Cai etc. at 3 ' end place at 5 ' end place; RNA 2004,10 (12), 1957-1966; Lee etc., Nature 2003,425 (6956), 415-419; Kim Nat.Rev.Mol.Cell.Biol.2005,6 (5), 376-385).Next procedure for through Exportin-5 (carrier protein) with precursor miRNA transport nucleus be transported in the Cytoplasm (Yi etc., Genes.Dev.2003,17 (24), 3011-3016, Bohnsack etc., RNA2004,10 (2), 185-191).The GTP combining form of the cofactor Ran of Exportin-5 and Exportin-5 is discerned 3 of 2 nucleotide ' outstanding together and it is combined (Basyuk etc. with the adjacent blades with precursor miRNA characteristic; Nucl.Acids Res.2003; 31 (22), 6593-6597, Zamore Mol.Cell.2001; 8 (6), 1158-1160).In Cytoplasm, the GTP hydrolysis causes discharging precursor miRNA, processes said precursor miRNA (Bohnsack etc.) by cellular endonuclease III enzyme Dicer subsequently.Dicer at first obtains identification because of its effect in the siRNA that produces mediate rna interference (RNAi).Dicer and its cofactor TRBP mating reaction (Transactivating region binding protein; Chendrimata etc., Nature2005,436 (7051), 740-744) and PACT (interferon-inducible double strand-RNA-dependant protein kinase activator; Lee etc., EMBO J.2006,25 (3), 522-532).These enzymes combine at 2 nucleotide highlights that are positioned at 3 of precursor miRNA hairpin structure base portion ' end; And the removal end-rings, thereby produce about 21-nt miRNA duplex intermedium that two ends all have the outstanding and 3 ' hydroxyl of 5 ' single phosphoric acid, 3 ' 2 nucleotide.Subsequently; Selection guides chain (5 ' end of this miRNA guiding chain is unstable on energy) to incorporate among the RISC (the inductive reticent complex of RNA) miRNA, discharges simultaneously and degraded " passerby " chain (Maniataki etc., Genes.Dev.2005; 19 (24), 2979-2990; Hammond etc., Nature 2000,404 (6775), 293-296).It is definite by halves that the compositions of RISC still keeps, but key component is the member (Maniataki etc. of Argonaute (Ago) protein families; Meister etc., Mol.Cell.2004,15 (2), 185-197).
Subsequently, ripe miRNA guides to complementary mRNA kind with RISC.If said target mrna has complete complementarity with the RISC that has a miRNA, will make so mRNA cracking and degraded (Zeng etc., Proc.Natl.Acad.Sci.USA 2003,100 (17), 9779-9784; Hutvagner etc., Science2002,297 (5589), 2056-2060).But as modal situation in mammalian cell, the miRNA targeting has the mRNA of incomplete complementarity and suppresses their translation, this cause the reduction of respective egg white matter express (Yekta etc., Science 2004,304 (5670), 594-596; Olsen etc., Dev.Biol.1999,216 (2), 671-680).5 of miRNA ' distinguishes (being called seed zone (seed region)); Especially very important concerning the miRNA targeting in fact in nucleotide 2 to 7 or the coupling between 8 miRNA of place and the target sequence (since the position 1 at 5 ' end place) of miRNA; And this seed coupling also becomes the key principle (Lewis etc. that are widely used in the computer forecast miRNA targeting; Cell 2005,120 (1), 15-20; Brennecke etc., PLoS Biol.2005,3 (3), e85).Main miRNA through a plurality of complementary site mediation miRNA-mRNA duplex among 3 ' UTR regulates, but has many exceptional cases.MiRNA also can combine the coding region of 5 ' UTR and/or mRNA, and this causes similar result, and (Lytle etc., Proc.Natl.Acad.Sci.USA 2007,104 (23), 9667-9672).
Ribonuclease H
Ribonuclease H for 3 of RNA in the DNA/RNA duplex '-O-P bond cleavage is separated with generate 3 '-hydroxyl and 5 '-ribonuclease of phosphate terminal product.Ribonuclease H is nonspecific Cobra venom endonuclease and passes through the cracking of hydrolysis mechanism by the bivalent metal ion auxiliary catalysis RNA of desmoenzyme.Almost (from archeobacteria and prokaryote to eukaryote) all found the member of ribonuclease H family in all organisms.During dna replication dna, think that ribonuclease H cuts the responsible RNA primer that causes the generation of Gang Qipianduan; Yet the ribonuclease H enzyme possibly more be usually used in any DNA:RNA hybridization sequences (for example, being generally the DNA:RNA hybridization sequences that in mammal length is 4 or more a plurality of base pairs) of cracking sufficient length.
Microrna and the treatment of Microrna appearance
Microrna (miRNA) is described as combining to work through 3 ' UTR with the template transcript, thus through disturbing relevant but different mechanism to suppress expression by template transcript encoded protein matter with typical RNA.Specifically, think that miRNA works through the translation of reduction target transcript rather than through reducing its stability.The length of natural miRNA is typically about 22nt.It is believed that they be derived from about 70nt long be called as hour preface RNA than larger precursor.
Such as siRNA (and more particularly; Such as miRNA) agent interfering tolerable siRNA/ template (miRNA/ template) duplex in the mispairing of greater number; And the especially mispairing in the tolerable duplex center; Said agent interfering is combined in 3 ' UTR (or other place in the target transcript is for example in the repeat element of the transcript of Notch and/or Notch family).In fact, proof on evidence, some mispairing possibly be expectation or required, show this type of mispairing continually such as natural stRNA, and demonstrated suppress external translation miRNA also be like this (Zeng etc., Molecular Cell, 9:1-20).For example, when hybridizing with the target transcript, this type of miRNA comprises isolating complementary two extensions fully by the mispairing district usually.It is right that this kind hybridization complex comprises complete complementary two districts (duplex part) and the single at least base mismatch that contain nucleotide pair usually, and this base pair possibly be (for example) G:A, G:U, G:G, A:A, A:C, U:U, U:C, C:C, G:-, A:-, U:-, C:-etc.This type of mispairing nucleotide, if especially series connection exists (for example, two, three or four nucleotide mispairing districts) can form protrusion, the duplex that this protrusion will be positioned on arbitrary side of this kind protrusion partly separates.Multiple structure is possible.For example, miRNA can comprise a plurality of nonidentities (mispairing) district.All comprise on the meaning of unpaired nucleotide at target and miRNA, nonidentity (mispairing) district needs not to be symmetric.For example, only described wherein the structure that a chain comprises unpaired nucleotide (Zeng etc., Figure 14).Usually, the length of complete complementary extension is 5 nucleotide at least in the miRNA, and for example length is 6,7 or more a plurality of nucleotide, and the length in mispairing district possibly be (for example) 1,2,3 or 4 nucleotide.
Usually; Any specific siRNA can play the effect of inhibition of gene expression through following approach: (i) " typically " siRNA approach; The stability of its transcript that hits reduces and is wherein preferably complementary fully between siRNA and the target usually; (ii) " alternate " approach (being characterized by the miRNA approach in the animal usually), the translation of its transcript that hits is suppressed.Usually, will be possible although single transcript can contain the district of the target that can be used as typical approach and alternative route through mechanism (i) with (ii) the transcript of institute's targeting will be different through mechanism by the transcript of specific siRNA institute targeting.(notice that term " typical case " and " substituting " are only used for convenience's sake, and be considered to reflect the historical timing of in zooblast, finding this type of mechanism usually, but do not reflect importance, effectiveness or the further feature of arbitrary mechanism).A common objective of siRNA design is the single transcript that has high degree of specificity through mechanism (i) targeting, and the effect of missing the target (comprising those effects that possibly (ii) cause through mechanism) is minimized.Yet; Specifically; The purpose of this invention is to provide the rnai agent of having a mind to have the mispairing residue; In order to simulate the active of natural miRNA or in order to produce the reagent to target RNA (the unknown at present corresponding miRNA to it), said rnai agent has the inhibition and/or the therapeutic efficiency/effectiveness of this type of " DmiRNA " agent that can tolerate this type of mispairing (and might be strengthened by this type mispairing certainly).
The miRNA agent should be with favourable and/or use miRNA based on the mode that " typical case " use of complete complementary siRNA (this siRNA works through machine-processed (i)) is expanded to the toleration of mispairing nucleotide (and be actually above mechanism existence (ii) in the cell and use naturally) suggestion.Because miRNA is natural molecule, so when using miRNA, have different advantages probably as therapeutic agent.MiRNA has benefited from For hundreds of millions of years and comes the evolution of their functions " fine setting ".Therefore, sequence-specific " missing the target " effect should not be the problem that under the situation of natural miRNA, exists, and should not be the problem that under the situation of the synthetic DmiRNA that is designed for the natural miRNA of direct modeling, exists of the present invention in the same way yet.In addition; MiRNA has evolved and has been the expression of the group of regulator gene; Thereby be in harmonious proportion downward modulation (in some situation, carrying out two kinds of functions at single miRNA simultaneously in to the cell of a plurality of target RNA hybridisms) in the driving, the result can accurately regulate complicated cell function.This kind replacement of natural miRNA with synthetic miRNA or miRNA analogies (for example can relate to; DmiRNA) introduce in the pathological tissues normal propagation, apoptosis, cell cycle and other cell function of attempting to recover to receive the downward modulation of one or more miRNA to influence.In some situation; The activation again that these miRNA regulate approach (has for example produced significant treatment response; In a research to cardiac hypertrophy; Cross by the adenovirus mediated miR-133 that sends generation of miRNA expression cassette that expressing watches for animals avoids the cardiac hypertrophy of agonist induction, yet the reduction of miR-133 causes the increase (Care etc., Nat.Med.13:613-618)) of loose markers in the wild-type mice that is produced by antagomir on the contrary.
Up to now, be accredited as in the people's gene group more than 600 miRNA and encoded, wherein expressed and processed this type of miRNA through the protein in nucleus and the Cytoplasm is made up.MiRNA is conservative and comprise about 2% of all mammalian genes at the vertebrates camber.Since each miRNA as if to regulate a plurality of (for example; Two, three, four, five, six, seven, eight, nine and even tens are to hundreds of) heterogeneic expression; So miRNA can play the effect of " master switch ", thereby regulate and coordinate a plurality of cellular pathways and process effectively.Through coordinating a plurality of expression of gene, miRNA plays a major role in fetal development, immunity, inflammation and cell growth and propagation.
Expression and functional study show that specific miRNA change of Expression is critical to various human diseases.More and more evidences show inducing favourable treatment response (Pappas etc., Expert Opin Ther Targets.12:115-27) in specific miRNA introducing disease cell and the tissue.Because some miRNA is as the obvious effect of tumor inhibitor, possible miRNA therapy will be hopeful to be used for cancer future most.Through following observation, can at least partly support to be used for the ultimate principle based on the miRNA therapy of (for example) cancer:
(1) when comparing with normal tissue, miRNA horizontal error regulation and control and expression in pathological tissues usually to change.Several researchs show the miRNA that changes level in (with respect to the corresponding normal tissue of cancerous tissue) cancerous tissue.Usually, the expression of change is the result of gene mutation, and said gene mutation causes specific miRNA to increase expression or reduces and express.Disease with unique miRNA expression characteristic can be used as diagnostic marker and prognostic markers thing, and available DsiRNA of the present invention (DmiRNA) reagent carries out targeting.
(2) mistake regulation and control miRNA is used for impelling cancer development through what play oncogene or tumor inhibitor.Oncogene is defined as the gene that its overexpression or improper activation cause tumor to form.Tumor inhibitor is for preventing that cell from becoming the required gene of cancerous cells; The downward modulation of tumor inhibitor or inefficacy are the common inducers of cancer.Two types gene is all represented preferred drug targets, because this targeting especially can work according to the molecular basis of particular cancers.The instance of carcinogenic miRNA is miR-155 and miR-17-92; Let-7 is the instance of tumor suppression miRNA.
(3) before clinical, in the zooscopy, use miRNA and come the inductive treatment response through blocking-up or reduction growth of tumor.Scientific literature provides Proof of Concept research, and this research proof is recovered the growth that the miRNA function can stop or be reduced in cancerous cell in external and the animal model.The instance of well-characterized is the anti-tumor activity to let-7 in the model of breast carcinoma and pulmonary carcinoma.DsiRNA of the present invention (DmiRNA) through being designed to simulate let-7 can be used for this type of cancer of targeting; And possibly use DsiRNA design parameter as herein described to produce to new DsiRNA (DmiRNA) reagent of target RNA with screening therapeutic lead compound; (for example there is not the natural miRNA of known correspondence to RNA at present for said; Repetition in Notch or other transcript), said new DsiRNA reagent is for for example reducing the reagent of tumor load in the clinical preceding animal model.
(4) given miRNA controls a plurality of cellular pathways, and therefore possibly have good therapeutic activity.Based on their biology, miRNA can play genomic " master switch ", thereby regulates a plurality of gene outcomes and coordinate a plurality of approach.The gene of being regulated by miRNA comprises the gene of traditional oncogene of coding and tumor inhibitor, manyly in the middle of them is elected to be drug targets individually by pharmacy industry.Therefore, through a plurality of genes relevant with disease and/or cancer of targeting, the miRNA therapy can have the activity of the lead compound that is superior to siRNA and other form.Regulate and control the early stage incident in the tumor generating process normally if observe the mistake of miRNA, the miRNA therapy of the miRNA of replacement disappearance so possibly be optimal therapy.
(5) miRNA is a natural molecule, and therefore is not easy to induce non-specific side effect.Evolution over millions of years helps to develop the regulating networks of miRNA, thus the interaction of fine setting miRNA and target messenger RNA.Therefore, when in suitable environment, using, miRNA and miRNA derivant (for example, through the natural miRNA of design simulation DmiRNA) will have any sequence-specific effect of " missing the target " hardly.
The physical property of siRNA and miRNA is similar.Therefore, the technology that is used to send siRNA (for example, DsiRNA of the present invention) effectively can be sent synthetic miRNA (for example, DmiRNA of the present invention) equally effectively.
The puting together and send of DsiRNA reagent
In certain embodiments, the present invention relates to be used to treat the method for suffering from disease or disease or being in the curee of development disease or disease risk.In this type of embodiment, DsiRNA can be used as the new therapeutic agent that is used for control disease or disease.This method comprises to patient's (for example, the people) uses pharmaceutical composition of the present invention, to reduce expression, level and/or the activity of target RNA.Even under the situation of said DsiRNA, also can reduce expression, level and/or activity through DsiRNA of the present invention by target RNA encoded polypeptides to the noncoding region (for example, target 5 ' UTR or 3 ' UTR sequence) of transcript.In view of their high degree of specificity, DsiRNA of the present invention is the target sequence of targeted cells and tissue specifically, and randomly with allele specific mode, wherein polymorphism allele is present in individuality and/or the colony.
When treatment disease or disease, can make DsiRNA contact curee's cell or tissue, for example, performance protein mistake regulation and control and/or otherwise by the cell or tissue of targeting with the curee that reduces protein level.For example, can make with target RNA sequence in the same basically DsiRNA of all or part in vivo or the external cells contacting of planting therewith, or said DsiRNA introduced in this kind cell.Similarly, can to the curee who suffers from disease or disease or be in development disease or disease risk directly use with target RNA sequence in the same basically DsiRNA of all or part.
The therapeutic use of DsiRNA reagent of the present invention relates to the preparation that uses DsiRNA reagent, and said DsiRNA reagent comprises a plurality of different DsiRNA reagent sequences.For example; Can with in the current said reagent two or more, three or more a plurality of, four or more a plurality of, five or combination such as more a plurality of be used to produce preparation, the for example preparation of a plurality of not same districts of targeting target RNA or the not only preparation of the relevant cellular targets gene of targeting target RNA but also targeting (for example) and disease or disease (RNA is relevant with target).Also can make up DsiRNA reagent of the present invention, so that arbitrary chain of DsiRNA reagent two or more districts of targeted rna target independently, perhaps make the cellular targets gene of a chain targeting said target mrna known in the art of DsiRNA reagent.
That uses the targeting target nucleic acid molecule can also provide effective inhibition of rna level and expression greater than the multi-functional DsiRNA molecule in a district.For example, single multi-functional DsiRNA construct of the present invention can targeting above both.
Therefore, DsiRNA reagent of the present invention can perhaps make up with other medicines individually or combine to be used for to treat, suppresses, alleviates or prevention and target RNA diseases associated or disease.For example, under the condition that is suitable for treating, can use (individually or with one or more drug regimens) said DsiRNA molecule to the curee or to other the suitable cell that it will be apparent to those skilled in the art.
The DsiRNA molecule can also be used for treating, suppress, alleviate or the target RNA diseases associated or the disease of prevention and curee or organism with the combination of other known treatment thing.For example, said DsiRNA molecule can be used for treating, suppress, alleviate or preventing curee known in the art or organism and target RNA diseases associated or disease with one or more known compounds, treatment or method combination.
DsiRNA reagent of the present invention can be puted together or not put together with another part (for example, non-nucleic acid moiety is such as peptide), organic compound (for example, dyestuff, cholesterol etc.) 5 ' end or 3 ' end place of the sense strand or the antisense strand of DsiRNA reagent (for example).Compare with corresponding unconjugated DsiRNA reagent, modify in this way DsiRNA reagent can improve cellular uptake or strengthen the cell-targeting of DsiRNA reagent derivant active, have the purposes of following the trail of the DsiRNA reagent derivant in the cell or compare the stability that improves DsiRNA reagent derivant with corresponding unconjugated DsiRNA reagent.
The dsRNA-peptide conjugate of further puting together with therapeutic agent is also contained in the present invention, and said therapeutic agent is for for example treating or improving the symptom of disease (for example, cancer) and/or the reagent of progress.
Introduce the method for nucleic acid, carrier and host cell
Can DsiRNA reagent of the present invention is direct (that is ground in the cell) introduce in cell; Perhaps with said DsiRNA reagent extracellularly introduce in the organism cavity, in the intercellular space, circulation, the said DsiRNA reagent of oral introducing maybe can be introduced said DsiRNA reagent through cell or organism are immersed in the solution that contains nucleic acid.Blood vessel or blood vessel outer circulation, blood or lymphsystem and cerebrospinal fluid are can nucleic acid be introduced site wherein.
Can use delivery of nucleic acids method as known in the art to introduce DsiRNA reagent of the present invention, said delivery of nucleic acids method comprises that injection contains the solution of nucleic acid, bombards, cell or organism are soaked in nucleic acid solution or have the electroporation that carries out cell membrane under the situation of nucleic acid through the microgranule by the nucleic acid parcel.Also can use other method known in the art that is used for nucleic acid is introduced cell, such as the transportation of the carrier transportation of lipid mediation, chemicals mediation and cationic-liposome transfection (such as, calcium phosphate) etc.Can nucleic acid and other one or more active other components below performance be introduced together: strengthen cell or to the picked-up of nucleic acid or otherwise improve inhibition target RNA.
Have target RNA cell can from germ cell line or somatic cell, all-round or polyenergic, division or not splitted, essence or epithelium, immortality or cell transformed etc.Cell can be the cell of stem cell or differentiation.The cell type of differentiation comprises adipose cell, fibroblast, myocyte, myocardial cell, endotheliocyte, neuron, neuroglia, hemocyte, megalokaryocyte, lymphocyte, macrophage, neutrophil cell, eosinophilic granulocyte, basophilic granulocyte, mastocyte, leukocyte, granulocyte, keratinocyte, chondrocyte, osteoblast, osteoclast, hepatocyte and endocrine gland or eccrine cell.
According to the dosage of particular target RNA sequence with the DsiRNA reagent material of being sent, this method can cause the RNA funtion part or completely lose.At least 50%, 60%, 70%, 80%, 90%, 95% or 99% or above target cell in the reduction or the forfeiture of rna level or expression (rna expression or encoded polypeptides express) be exemplary.Suppress rna level or expression and refer to that there be not (or observable reduction) in RNA or RNA encoded protein level.Specificity refer to suppress RNA and in the pair cell other gene do not have the ability of obvious influence.Suppress the result and can confirm that said biochemical technology such as RNA solution hybridization, nuclease protection, Northern hybridization, reverse transcription, use microarray carry out the cell analysis (FACS) of gene expression monitoring, antibodies, ELISA (ELISA), western blotting, radioimmunoassay (RIA), other immunoassay and fluorescence-activation through the external characteristic of check cell or organism or through biochemical technology.Also can measure the inhibition of DsiRNA reagent of the present invention based on using the effect of this kind DsiRNA reagent to the generation/progress of disease or disease (no matter be in the body or external) to target RNA sequence; Said disease or disease are relevant with target RNA, for example tumor formation, growth, transfer etc.Treatment and/or reduction tumor or cancerous cell level can comprise the growth that stops tumor or cancerous cell or reduce tumor or cancerous cell level; Make tumor or cancerous cell level reduce (for example) 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% or more than; And can for example can realize that the cancerous cell level is reduced to 1/10,1/100,1/1000,1/10 with logarithm term tolerance through use DsiRNA reagent of the present invention to cell, tissue or curee 5, 1/10 6, 1/10 7
The inhibition of the RNA mediation in pair cell system or the whole organism can be measured the reporter gene that protein is easy to measure or the expression of drug resistance gene.This kind reporter gene comprises: acetohydroxy acid synthase (AHAS), alkali phosphatase (AP), beta galactosidase (LacZ), β glycuronidase (glucoronidase) (GUS), chloramphenicol acetyltransferase (CAT), green fluorescent protein (GFP), horseradish peroxidase (HRP), luciferase (Luc), nopaline synthase (NOS), octopine synthase (OCS) and derivant thereof.Can use the multiple choices labelling that to give ampicillin, bleomycin, chloromycetin, gentamycin, ST-4331, kanamycin, lincomycin, methotrexate, careless fourth phosphine, puromycin and tetracyclin resistance.According to mensuration, the inhibition degree is measured in the quantitative permission of gene expression amount, this inhibition degree is higher by 10% than the cell of not handling according to the present invention, 33%, 50%, 90%, 95% or 99%.
May cause the inhibition in the cell (for example, at least 10%, 20%, 50%, 75%, 90% or 95% target cell) in less part than the injection mass of low dosage and the time after the reticent agent of prolongation administering RNA.Similar amount of suppression under the level that quantitatively can be presented at accumulation target RNA or translation target proteins of gene expression in the cell.For example, suppressing efficient can be through the quantitative determination of gene outcome in the assessment cell; Can be used on the hybridization probe that has nucleotide sequence outside the district that is used to suppress DsiRNA and detect RNA, or the polypeptide of the available antibody test translation that produces to the peptide sequence in this district.
Should send the amount of at least one copy to introduce DsiRNA reagent to allow each cell delivery.Higher dosages of substance (for example, each cell at least 5,10,100,500 or 1000 copies) can produce more effective inhibition; Also can be used for application-specific than low dosage.
Pharmaceutical composition
In certain embodiments, the present invention provides the pharmaceutical composition that comprises dsRNA-peptide reagent of the present invention.Can suitably prepare dsRNA-peptide reagent sample, and can allow the sample entering cell of enough parts dsRNA-peptide reagent sample to be introduced in the cellular environment with any way of induced gene reticent (if generation).Many preparations of dsRNA are known in the art and can use, as long as dsRNA can get into target cell so that dsRNA can work.For example, referring to U.S.'s publication application 2004/0203145A1 number and 2005/0054598A1 number.For example, dsRNA-peptide reagent of the present invention can be prepared in buffer solution (such as the normal saline solution of phosphate-buffered), liposome, capsule structure and capsid.Preparation with DsiRNA reagent of cation lipid can help the transfection of DsiRNA reagent to cell.For example, can use cation lipid, such as lipofectin (United States Patent (USP) the 5th, 705, No. 188); The cation glycerol derivatives; And the polycation molecule, such as polylysine (disclosed PCT International Application No. WO 97/30731).Suitable lipid comprises: Oligofectamine, Lipofectamine (Life Technologies), NC388 (Ribozyme Pharmaceuticals; Inc.; Boulder, Colo) or FuGene 6 (Roche), all these lipids all can use according to the explanation of manufacturer.
This kind compositions comprises nucleic acid molecules and pharmaceutically acceptable carrier usually.As used herein term " pharmaceutically acceptable carrier " comprises saline, solvent, disperse medium, coating, antibacterial and antifungal, isotonic agent and the absorption delay agent etc. that are fit to medicament administration.The reactive compound that replenishes also can be incorporated in the compositions.
The compounding pharmaceutical compositions makes the route of administration that is suitable for expecting.The instance of route of administration comprises parenteral administration, for example intravenous, Intradermal, subcutaneous, oral (for example, sucking), percutaneous (part), through mucous membrane and rectal administration.The solution or the suspension that are used for parenteral, Intradermal or subcutaneous application can comprise following component: sterile diluent, such as water for injection, normal saline solution, fixed oil, Polyethylene Glycol, glycerol, propylene glycol or other synthetic; Antibacterial is such as benzyl alcohol or methyl parahydroxybenzoate; Antioxidant is such as ascorbic acid or sodium sulfite; Chelating agen is such as ethylenediaminetetraacetic acid; Buffer is such as acetate, citrate or phosphate; And the reagent that is used for adjustment of tonicity, such as sodium chloride or glucose.Usable acid or alkali (such as, hydrochloric acid or sodium hydroxide) adjusting pH.Can parenteral administration be enclosed in ampoule, disposable syringe or the multidose bottle of being processed by glass or plastics.
The pharmaceutical composition that is suitable for injecting use comprises aseptic aqueous solution (under the water miscible situation) or dispersant and the sterilized powder that is used for temporarily preparing sterile injectable solution or dispersant.Concerning intravenous administration, suitable carriers comprise normal saline, bacteriostatic water, Cremophor EL.TM. (BASF, Parsippany, N.J.) or the normal saline of phosphate-buffered (PBS).In all cases, compositions must be aseptic and should be liquid, so that injection.Compositions should produce with storage condition under stablize, and must avoid microorganism (such as, antibacterial and fungus) contamination and preserve.Carrier can be for containing the for example solvent or the disperse medium of water, ethanol, polyhydric alcohol (for example, glycerol, propylene glycol and liquid polyethylene ethylene glycol etc.) and suitable mixture thereof.For example, can through use coating (such as, lecithin), under the situation of dispersant through keeping required particle diameter, and, keep suitable flowability through using surfactant.Can realize preventing action of microorganisms through various antibacterial and antifungal (for example, p-Hydroxybenzoate, methaform, phenol, ascorbic acid, thimerosal etc.).Under many circumstances, preferably in compositions, comprise isotonic agent, for example sugar, polyhydric alcohol (such as, mannitol, Sorbitol), sodium chloride.Can be through in compositions, comprising the absorption that the reagent (for example, aluminum monostearate and gelatin) that postpones to absorb can prolong injectable compositions.
Can through the reactive compound of aequum and the combination of a kind of mentioned component or mentioned component are incorporated in the appropriate solvent, carry out filtration sterilization subsequently as required, prepare sterile injectable solution.Usually, prepare dispersant through reactive compound is incorporated in the aseptic vehicle, said aseptic vehicle contains alkaline disperse medium and from required other composition in above-mentioned those compositions.Under the situation of the sterilized powder that is used to prepare sterile injectable solution, preferred manufacturing procedure is vacuum drying and lyophilization, this method obtain containing active component with from it before through the powder of any other composition of wanting of the solution of aseptic filtration.
Orally administered composition generally includes inert diluent or edible carrier.Purpose from oral medication property is used can merge reactive compound and excipient, and use with the form of tablet, lozenge or capsule (for example, gelatine capsule).Can also use liquid-carrier, prepare Orally administered composition as collutory.Can comprise pharmaceutically compatible binding agent and/or Adjuvanting material a part as compositions.Tablet, pill, capsule, lozenge etc. can contain following the have composition of similarity or in the chemical compound any: binding agent, such as microcrystalline Cellulose, Tragacanth or gelatin; Excipient is such as starch or lactose; Disintegrating agent is such as alginic acid, Primogel or corn starch; Lubricant is such as magnesium stearate or Sterotes; Fluidizer is such as silica sol; Sweeting agent is such as sucrose or glucide; Or flavoring agent, such as Herba Menthae, methyl salicylate or orange flavor flavoring agent.
For using through suction, chemical compound is to send with aerosol spray form, and this aerosol is from pressure vessel or contain suitable propellant (for example, gas is like carbon dioxide) allotter, or aerosol apparatus.These methods comprise United States Patent(USP) No. 6,468, those described in 798.
Can also carry out systemic administration through through mucous membrane or percutaneous mode.Concerning through mucous membrane or applied dermally, in preparation, can use the penetrating agent that is suitable for penetration barriers.This type of penetrating agent is normally known in the art, and comprises detergent, bile salts and the fusidic acid derivatives that for example is suitable for mucosal administration.Can accomplish mucosal administration through using nose spraying or suppository.Concerning applied dermally, reactive compound preparation become be generally ointment known in the art, ointment, gel or emulsifiable paste.
Can also compound be become suppository (for example, comprising conventional suppository base, such as cocoa butter and other glyceride) or be used for the form of the enema,retention that rectum sends.
Can also use methods known in the art to come administered compound through transfection or infection, said method includes but not limited to McCaffrey etc., (2002), Nature, 418 (6893), 38-9 (hydrodynamics transfection); Xia etc., (2002), Nature Biotechnol, 20 (10), 1006-10 (virus-mediated sends); Or Putnam (1996), Am.J.Health Syst.Pharm.53 (2), 151-160, erratum at Am.J.Health Syst.Pharm.53 (3), the method described in 325 (1996).
Can also come administered compound through any method that is suitable for administration of nucleic acid reagent (such as, dna vaccination).These methods comprise particle gun, biological syringe and skin patch; And do not have needle method, such as United States Patent (USP) the 6th, 194; Disclosed microgranule dna vaccination technology in No. 389; And like United States Patent (USP) the 6th, 168, the mammal percutaneous needleless vaccination of disclosed use powder type vaccine is technological in No. 587.In addition, intranasal delivery also is possible, especially like Hamajima etc., (1998), and Clin.Immunol.Immunopathol., 88 (2), described in the 205-10.Can also use liposome (for example, like United States Patent (USP) the 6th, 472, described in No. 375) and microencapsulated technology.But can also use the microgranule delivery system (for example, like United States Patent (USP) the 6th, 471, described in No. 996) of biodegradable targeting.
In one embodiment, use will protect chemical compound to avoid the preparing carriers reactive compound of from health, eliminating fast, and said carrier comprises implant and micro-encapsulated delivery system such as the sustained release preparation.Can use biodegradable, biocompatible polymer, such as ethylene vinyl acetate, gather anhydride, polyglycolic acid, collagen protein, poe and polylactic acid.Can use this type of preparation of standard technique preparation.Can also be from Alza Corporation and Nova Pharmaceuticals, Inc. is purchased these materials.Liposome suspension (comprising the liposome that has to the targeting infected cell of the monoclonal antibody of virus antigen) also can be used as pharmaceutically acceptable carrier.Can prepare these materials according to those skilled in the art's known method (for example, like United States Patent (USP) the 4th, 522, the method for being described in No. 811).
Can for example be used to measure LD50 (making 50% the fatal dosage of colony) and ED50 (to the effective dosage of 50% mass treatment) through the toxicity and the therapeutic efficiency of this compounds of standard drug program determination in cell culture or laboratory animal.Dosage rate between toxicity and the therapeutical effect is a therapeutic index, and said dosage rate can be expressed as ratio LD50/ED50.The chemical compound that preferably presents higher therapeutic index.Though can use the chemical compound that presents toxic side effects, should note designing the delivery system at the position that makes this type of targeting compounds affected tissue, so that the potential damage of non-infected cells is minimized, and then reduce side effect.
Can be used to prepare the dosage range that supplies the people to use from the data of cell culture assays and zooscopy acquisition.The dosage of this compounds preferably has few or is not having in the toxic circulation composition scope (this concentration range comprises ED50).Dosage can depend on used dosage form and used route of administration and in this scope, change.Concerning any chemical compound that is used for method of the present invention, can from cell culture assays, treat effective dose according to a preliminary estimate.Can in animal model, prepare dosage, to reach the IC that comprises as being measured in the cell culture 50The circulating plasma concentration range of (that is, reaching the concentration of the test compounds of maximum half inhibition of symptom).This kind information can be used for confirming more accurately to be used for people's dosage.For example, can pass through the high-efficient liquid phase chromatogram technique measuring blood plasma level.
Such as among this paper definition, the treatment effective dose of nucleic acid molecules (that is effective dose) depends on selected nucleic acid.For example, if select the plasmid of encoding D siRNA reagent, the scope of can using so is approximately the single dose of 1pg to 1000mg; In some embodiments, can use 10pg, 30pg, 100pg or 1000pg, perhaps 10ng, 30ng, 100ng or 1000ng, perhaps 10 μ g, 30 μ g, 100 μ g or 1000 μ g, perhaps 10mg, 30mg, 100mg or 1000mg.In some embodiments, can use 1 to 5g compositions.Compositions can once-a-day administration or is repeatedly used to using one or many (comprising every other day once) weekly.It will be appreciated by those skilled in the art that some factor may influence for effectively treating required dosage and the time-histories of curee, said factor includes but not limited to the seriousness of disease or disease, previous treatment, curee's basic health and/or other disease of age and existence.In addition, use protein, polypeptide or the Antybody therapy curee of treatment effective dose can comprise single therapy, or preferably, can comprise a series of treatments.
Such as preceding text definition; Because according to the targeting of peptide useful in the treatment of the present invention " raising " with the conjugate of peptide-dsRNA; So with do not put together and compare for the amount or the dosage that reach the required identical dsRNA of combination, association or the internalization of peer-level with peptide; Need less dsRNA (than the dsRNA of low dosage), this is by the IC in hereinafter described measuring 50Confirm.For example, as in the body or external measured, with the IC of the identical dsRNA that does not put together with peptide 50Compare, reach the IC that 50% of RNA/ gene expression reduces required dsRNA-peptide conjugate 50Reduce (for example, referring to Hefner etc., J Biomol Tech.2008 JIUYUE: 19 (4) 231-237; Zimmermann etc., Nature.2006 May 4: 441 (7089): 111-114; Durcan etc., the Mol Pharm.2008 7-8 month; 5 (4): 559-566; Heidel etc., Proc Natl Acad Sci U S A.2007: 104 (14): 5715-5721).
Such as among this paper definition; The useful dosage of dsRNA-peptide is about 0.1mg/kg to 100mg/kg, for example 0.2kg/mg to 50kg/mg, 0.5kg/mg to 30kg/mg or 0.5mg/kg to 20mg/kg (comprise 0.5kg/mg, 0.6kg/mg, 0.7kg/mg, 0.8kg/mg, 0.9kg/mg, 1kg/mg, 1.5kg/mg, 2kg/mg, 2.5kg/mg, 3kg/mg, 3.5kg/mg, 4kg/mg, 4.5kg/mg, 5kg/mg, 5.5kg/mg, 6kg/mg, 6.5kg/mg, 7kg/mg, 7.5kg/mg, 8kg/mg, 8.5kg/mg, 9kg/mg, 9.5kg/mg, 10kg/mg, 10.5kg/mg, 11kg/mg, 11.5kg/mg, 12kg/mg, 12.5kg/mg, 13kg/mg, 13.5kg/mg, 14kg/mg, 14.5kg/mg, 15kg/mg, 15.5kg/mg, 16kg/mg, 16.5kg/mg, 17kg/mg, 17.5kg/mg, 18kg/mg, 18.5kg/mg, 19kg/mg, 19.5kg/mg, 20kg/mg or more than).
For example; Can use methods known in the art (to include but not limited to preceding text Xia etc.; (2002) those methods described in), nucleic acid molecules of the present invention is inserted in the expression construct (for example, viral vector, retroviral vector, expression cassette or plasmid viral vector).For example, can through suck, oral, intravenous injection, local application be (referring to United States Patent (USP) the 5th, 328; No. 470) or through stereotactic injection (for example referring to, Chen etc., (1994); Proc.Natl.Acad.Sci.USA, 91,3054-3057) expression construct is delivered to the curee.The pharmaceutical preparation of delivery vector can comprise the carrier in the acceptable diluent, maybe can comprise the sustained-release matrix that wherein is embedded with delivery vehicle.Perhaps, under the situation that complete delivery vector can intactly be generated from reconstitution cell (for example, retroviral vector), pharmaceutical preparation can comprise the one or more cells that produce genes delivery system.
Expression construct can be any construct that is applicable in the suitable expression system, and includes but not limited to retroviral vector known in the art, linear expression cassette, plasmid and virus or virogeny carrier.This type of expression construct can comprise one or more inducible promoters, RNA Pol III promoter systems (such as, U6snRNA promoter or H1RNA polymerase III promoter) or other promoter known in the art.Construct can comprise a chain or two chains among the siRNA.The expression construct of expressing two chains can also comprise the circulus that connects two chains, or every chain can be transcribed by intravital independent startup of identical structure independently.Every chain can also (for example, Tuschl) be transcribed (2002, Nature Biotechnol 20:500-505) by the individual tables expression constructs.
Can recognize, the method in the DsiRNA reagent introducing cellular environment will be depended on the environment composition of cell type and cell.For example, when in liquid, finding cell, a kind of preferred preparation be for example in lipofectamine with the preparation of lipid formulations, and can DsiRNA reagent be added directly to the liquid environment of cell.For example, can also pass through intravenous, intramuscular or peritoneal injection or oral, perhaps, lipid formulations used to animal through sucking or other method known in the art.When preparation is suitable for when using in the animal (such as, mammal, and more particularly being the people), preparation also is pharmaceutically acceptable.The pharmaceutically acceptable preparation that is used to use oligonucleotide is known and can uses.In some instances, possibly preferably in buffer or saline solution, prepare DsiRNA reagent, and the DsiRNA reagent of preparation is directly injected cell, in the research of using oocyte.Also can carry out the direct injection of DsiRNA reagent duplex.To introducing the appropriate method of dsRNA (for example, DsiRNA reagent), referring to U.S.'s publication application 2004/0203145A1 number.
Must introduce the DsiRNA reagent of appropriate amount, and this tittle can use standard method to confirm by rule of thumb.Usually, the valid density of single DsiRNA reagent type in cellular environment is about 50 nanomoles or following, and 10 nanomoles or following can working concentration be about 1 nanomole or following compositions perhaps.In another embodiment, working concentration is about 200 picomoles or following, 100 picomoles or following, 50 picomoles or following, 20 picomoles or following and concentration even is that 10 picomoles or following, 5 picomoles or following, 2 picomoles or following or 1 picomole or following method can be used for many environment.
Can carry out said method through the DsiRNA reagent composition is added in any extracellular matrix that can make cell survival, condition is that preparation DsiRNA reagent composition is so that the DsiRNA reagent of q.s can get into cell to bring into play its effect.For example, this method be applicable to be present in liquid (such as, liquid culture or cell growth medium) in, organize in the explant or complete organism (comprising animal, such as, mammal, and especially is the people) in cell.
The level of RNA or active can mensuration by this area any appropriate method existing known or exploitation afterwards.Can recognize that the method that is used to measure target RNA and/or target rna expression can be depending on the character of target RNA.For example, under the proteinic situation of target RNA sequential coding, term " expression " can refer to be derived from the protein or the RNA/ transcript of target (genomic or the external source source) gene.In this type of situation, the expression of target RNA can be through the amount of directly measuring target RNA/ transcript or the quantitative determination of passing through the protein of measurement target RNA.Protein can be in protein determination, and (such as through staining or immunoblotting) measures, if the perhaps measurable reaction of protein catalysis can be measured protein through measuring reaction rate so.All these class methods are known in the art and can use.Measure in plan under the situation of target rna level, can use the art-recognized any method (for example, RT-PCR, RNA blotting etc.) that is used to detect rna level.When using DsiRNA reagent targeted rna of the present invention; Estimate also that DsiRNA reagent reduces curee, tissue, cell (external or body in) or in cell extract the measurement of the effect of RNA or protein level can also be used for (for example confirming the phenotype relevant with the specific RNA of target; Disease or disease, for example cancer or tumor formation, growth, transfer, diffusion etc.) the reduction degree.Any above-mentioned measurement can be carried out on cell, cell extract, tissue, tissue extract or any other suitable source material.
Can confirm whether the expression of target RNA reduces through any appropriate method that can detect the rna level variation reliably.Usually, through indigested DsiRNA is introduced in the cellular environment,, measure the level of target RNA subsequently and confirm so that at least a portion in the said DsiRNA reagent gets into Cytoplasm.On identical untreated cell, carry out identical measurement, and the result who relatively from each is measured, obtains.
Can DsiRNA reagent be formulated as pharmaceutical composition, this pharmaceutical composition comprises DsiRNA reagent and the pharmaceutically acceptable carrier that the pharmacology goes up effective dose.Pharmacology or treatment effective dose refer to that DsiRNA reagent produces the pharmacology of expection, therapeutic or preventative result's amount effectively.Phrase " pharmacology's effective dose " and " treatment effective dose " or only " effective dose " refer to the amount that RNA produces pharmacology, therapeutic or the preventative result of expection effectively.For example, if when relevant with disease or disease measurable parameter reduces at least 20%, think that given clinical treatment is effective, the treatment effective dose that is used to treat the medicine of this disease or disease so is for making that parameter reduce at least 20% required amount.
Can pass through any way known in the art; Use the pharmaceutical composition of suitable preparation of the present invention; Such as through the parenteral path, it comprises that intravenous, intramuscular, intraperitoneal, subcutaneous, percutaneous, respiratory tract (aerosol), rectum, vagina and part (comprising oral cavity and Sublingual) use.In some embodiments, through intravenous or intraperitoneal transfusion or injection drug administration compositions.
Usually, the proper dosage unit of dsRNA is within receiver's's every day per kilogram of body weight 0.001 to 0.25 nanogram range or in every day per kilogram of body weight 0.01 to 20 microgram scope or in every day per kilogram of body weight 0.01 to 10 microgram scope or in every day per kilogram of body weight 0.10 to 5 microgram scope or in every day per kilogram of body weight 0.1 to 2.5 microgram scope.The pharmaceutical composition that can once-a-day administration comprises dsRNA.Yet, therapeutic agent also can with contain 2,3,4,5,6 or the dosage unit of more sub-doses in one day by suitable interval administration.In the case, contained dsRNA must correspondingly reduce in each sub-doses, to reach total daily dose unit.For example, can also use traditional extended release preparation (this delivery formulations be provided at continue in some days periods and as one man discharge dsRNA), dosage unit is compound to be used for the single dose in some days.Extended release preparation is well known in the art.In this embodiment, dosage unit contains corresponding a plurality of daily dose.No matter preparation how, pharmaceutical composition must contain the dsRNA of the quantity of expression of target gene among the animal or human who is enough to suppress to be treated.The compound mode of compositions should make the dsRNA summation together of a plurality of units contain enough dosage.
Can from cell culture assays and zooscopy, obtain data, be suitable for people's suitable dosage ranges with formulation.The dosage of compositions of the present invention should comprise ED 50In the circulation composition scope of (as measuring), wherein have few toxicity or do not have toxicity by known method.The path of using of depending on dosage form and use, dosage can change in this scope.Concerning any chemical compound that is used for method of the present invention, can from cell culture assays, treat effective dose according to a preliminary estimate.Can in animal model, formulate dosage, to reach like the determined IC of comprising in cell culture 50The circulating plasma concentration range of (that is, realizing the concentration of the test compounds of maximum half inhibition of symptom).This kind information can be used for confirming more accurately to be used for people's dosage.The level of can standard method (for example, through HPLC) measuring dsRNA in the blood plasma.
Can pharmaceutical composition be included in test kit, container, packing or the liquor separator with using explanation.
Therapeutic Method
The present invention provides the curee's of the risk that treatment is in disease or disease (or to disease or disease responsive) preventative and therapeutic method; Said disease or disease are completely or partially by target RNA (for example; The mistake regulation and control and/or the raising of transcript and/or protein level) cause, maybe can be through selectivity targeting target RNA treatment.
As used herein; " treatment " be defined as to suffer from disease or disease, have the symptom of disease or disease or have the patient of development disease or disease tendency to use or the administering therapeutic agent (for example; DsiRNA reagent or carrier or its transgenic of encoding); Perhaps to using or the administering therapeutic agent, with the symptom that reaches treatment, cure, alleviate, alleviate, change, remedy, improve, improve or influence this disease or disease, this disease or disease or the purpose of ill tendency from this patient's isolated tissue or cell line.
In one aspect; The present invention is provided for through preventing the curee to suffer to curee's administering therapeutic agent (for example, DsiRNA reagent or carrier or its transgenic of encoding) to state the method for disease or disease (comprising that (for example) prevents transformation event takes place through the expression that suppresses target RNA in the curee).Having a kind of in can be for example measuring through diagnosis described herein or prediction of the curee of the risk of the disease of taking a disease or its to make up differentiates.Preventive can be used before the symptom characteristic performance of (for example) cancer that detects the curee or disease or disease, to prevent this disease or disease, perhaps delayed its development.
Another aspect of the present invention relates to the method that is used for therapeutic ground (that is, changing the outbreak of the symptom of disease or disease) treatment curee.Can external (for example, through cell is cultivated with DsiRNA reagent) or in vivo (for example, through use DsiRNA reagent to the curee) carry out these methods.
About preventative and therapeutic treatment method, can adjust or revise this type of treatment specially based on the knowledge that from the pharmacogenomics field, obtains.As used herein, " pharmacogenomics " refer to genome-based technologies (such as, gene sequencing, statistical genetics and gene expression analysis) be applied in clinical development and the medicine for sale.More particularly, said term relates to the gene of studying the patient and how to confirm its reaction to medicine (for example, patient's " drug reaction phenotype " or " drug response genotype ").Therefore, another aspect of the present invention is provided for using according to the drug response genotype of individuality the method for the preventative or therapeutic treatment of target RNA molecule of the present invention or target RNA regulator adjustment individuality.Pharmacogenomics allows clinicist or doctor physician that preventative or therapeutic treatment targeting can be obtained the patient of maximum benefit from this treatment, and avoids the patient that will stand the drug toxicity related side effects is treated.
Therapeutic agent can be tested in appropriate animal model.For example, DsiRNA reagent as described herein (perhaps encode its carrier or transgenic) can be used for effect, toxicity or the side effect of animal model to confirm to use said reagent to treat.Perhaps, reagent (for example, therapeutic agent) can be used for animal model to confirm the mechanism of action of this kind reagent.
Be used to assess the model of mRNA level and down-regulated expression
Cell culture
For example, can use the interior lytic activity of body of following program test dsRNA-peptide reagent of the present invention.
Can use HeLa cell or other mammalian cell in cell culture, to test dsRNA-peptide reagent of the present invention, to confirm the inhibition degree of target RNA and target protein.Select dsRNA-peptide reagent (for example, referring to Fig. 1 and said structure) to target as herein described.These reagent are delivered to that the HeLa cell cultivated in (for example) culture or other have transformed through suitable transfection agents or the mammalian cell of unconverted after, measure target RNA and suppress.Use the PCR in real time of amplification (for example to monitor; ABI 7700
Figure BDA0000133674250002361
), measure the relative quantity of target RNA with respect to actin or other suitable contrast.With to incoherent target; Or substituted at random randomization DsiRNA contrast, or compare to the suitable vehicle processing or the oligonucleotide sequence mixture of untreated tester simply to having identical total length and chemical constitution but in each position.Select the leading reagent of first and secondary, and be optimized to target.After selected optimum transfection agents concentration, use leading DsiRNA molecule to carry out RNA and suppress time course.
Figure BDA0000133674250002362
of mRNA (PCR in real time amplification monitoring) and Lightcycler are quantitative
After dsRNA sends, for example be used for the extensive Ambion Rnaqueous4-PCR purification kit that extracts or be used for the Ambion Rnaqueous-96 purification kit that 96 holes are measured, from the total RNA of cell preparation.Concerning Taqman analyzes, reporter gene dyestuff FAM that use (for example) is covalently bound at 5 ' end place or VIC and the quencher dyestuff TAMARA that puts together with 3 ' end, synthetic double labeling probe.Use 50 μ L reactants on (for example) ABI PRISM 7700 sequential detectors, to carry out one step RT-PCR amplification, said 50 μ L reactants comprise the total RNA of 10 μ L, 100nM forward primer, 100mM reverse primer, 100nM probe, 1xTaqMan PCR reaction buffer (PE-Applied Biosystems), 5.5mM MgCl 2, each 100uM dATP, dCTP, dGTP and dTTP, 0.2U ribonuclease inhibitor (Promega), 0.025U AmpliTaq Gold (PE-Applied Biosystems) and 0.2U M-MLV reverse transcriptase (Promega).Thermal cycle conditions can be included in 48 ℃ following 30 minutes, 95 ℃ following 10 minutes, carried out 40 circulations in following 15 seconds and carried out 40 circulations in following 1 minute at 95 ℃ subsequently at 60 ℃.Target KRAS mRNA level quantitatively be to measure with respect to reference material, this reference material is produced by total cell RNA of serial dilution (300,100,30,10ng/rxn) and to the for example normalized reference material of 36B4mRNA in parallel or same pipe TaqMan reaction.
Western blotting
Can use the micropreparative technique of standard to prepare nuclear extract (for example, referring to Andrews and Faller,, Nucleic Acids Research, 19,2499 in 1991).For example, use the protein extract of TCA deposition preparation from supernatant.Add the 20%TCA of equal volume to cell conditioned medium liquid, hatched on ice 1 hour, and through making its deposition in centrifugal 5 minutes.Washing precipitation in acetone, drying and in water, carry out resuspending.On 10%Bis-Tris NuPage (nuclear extract) or 4-12%Tris-glycine (supernatant extract) PAAG, make the cell protein extract run glue and said cell protein extract is transferred on the NC Nitroncellulose film.Can hatch 1 hour through (for example) and 5% skimmed milk, 4 ℃ of following and anti-hatching 16 hours, block non-specific binding subsequently.After washing, at room temperature use (1: 10,000 dilution) two and resist 1 hour, and use SuperSignal reagent (Pierce) detection signal.
In several cell culture systems, shown cation lipid can increase cell in the culture to the bioavailability of oligonucleotide (Bennet, etc., 1992, Mol.Pharmacology, 41,1023-1033).In one embodiment, DsiRNA molecule of the present invention and cation lipid form complex, to be used for cell culture experiments.DsiRNA and cation lipid mixture are in serum-free DMEM, to prepare before adding cell being about to.The DMEM doping is warming up to room temperature (about 20 to 25 ℃), and cation lipid is added to final desired concn and makes solution short time vortex.The DsiRNA molecule is added to final desired concn and makes solution short time vortex once more, and at room temperature hatched 10 minutes.In dose response experiments, after hatching 10 minutes, with the serial dilution of RNA/ lipid complex in DMEM.
Animal model
The effect of assessment dsRNA-peptide reagent is the important prerequisite of people's clinical trial in animal model.Various animal models like cancer known in the art and/or proliferative disease, condition of illness or disease go for the effect of clinical preceding assessment DsiRNA compositions of the present invention when regulating expression of target gene according to therapeutic use.
For example, if target is KRAS, as in the cell culture model, the responsive mouse tumor xenograft of so most Ras derives from expresses the proteic cancerous cell of sudden change Ras.Carrying H-Ras, to transform the nude mice antagonism Ras antisensenucleic acids of transitional cell bladder carcinoma cell line xenograft responsive, thus after the treatment period on the 31st, can suppress 80% tumor growth (Wickstrom, calendar year 2001, Mol.Biotechnol., 18,35-35).Zhang etc., 2000, Gene Ther., 7,2041 have described the anti-KRAS ribozyme adenopathy toxicity carrier (KRbz-ADV) of targeting KRAS mutant, and (KRAS codon 12GGT becomes GTT; Be respectively H441 cell and H1725 cell).Compare with the NSCLC H1650 cell that lacks relevant sudden change, express the sudden change proteic non-small cell lung cancer cell of KRa (NSCLC H441 and H1725 cell) and be used for the bare mouse different species graft.Use KRbz-ADV to treat in advance and can eliminate H441 and H1725 cellular transplant fully, and compare with 100% graft and tumor growth in the animal of untreated tumor cell of reception or control vector.The other mouse model (for example, at Kim etc., among the Cell 121:823-835, this has differentiated the effect of KRAS in promoting adenocarcinoma of lung) of KRAS mistake regulation and control/sudden change has also been described.Above-mentioned research proof suppresses Ras expression (for example, KRAS expresses) by anti-Ras reagent can suppress the tumor growth in the animal.
Therefore, these models can be used to assess the effects such as development of DsiRNA molecules in inhibiting KRAS level of the present invention, expression, the formation of lesion/cancer disease, growth, diffusion, other phenotype relevant with KRAS, disease or disease.These models and other model can be used for assessing the safety/toxicity and the effect of DsiRNA molecule of the present invention environment before clinical similarly.
Only if statement is arranged in addition, otherwise the routine techniques that embodiment of the present invention adopts chemistry, molecular biology, microbiology, recombinant DNA, hereditism, immunology, cytobiology, cell culture and genetically modified organism to learn, these technology are all in the skill of this area.For example, referring to Maniatis etc., 1982, Molecular Cloning (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.); Sambrook etc., 1989, Molecular Cloning, the 2nd edition (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.); Sambrook and Russell, 2001, Molecular Cloning, the 3rd edition (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.); Ausubel etc., 1992, Current Protocols in Molecular Biology (John Wiley&Sons comprises regular update); Glover, 1985, DNA Cloning (IRL Press, Oxford); Anand, 1992, Guthrie and Fink, 1991, Harlow and Lane, 1988, Antibodies, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.); Jakoby and Pastan, 1979, Nucleic Acid Hybridization (B.D.Hames&S.J.Higgins compiles, 1984); Transcription And Translation (B.D.Hames&S.J.Higgins compiles, 1984) Culture Of Animal Cells (R.I.Freshney, Alan R.Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B.Perbal, APractical Guide To Molecular Cloning (1984); Paper, and Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J.H.Miller and M.P.Calos compile, and 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, the 154th volume and the 155th volume volumes such as () Wu, Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker volume, Academic Press, London, 1987); Handbook Of Experimental Immunology, I-IV volume (D.M.Weir and C.C.Blackwell compile, 1986); Riott, Essential Immunology, the 6th edition, Blackwell Scientific Publications, Oxford, 1988; Hogan etc., Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986); Westerfield, M., The zebrafish book.A guide for the laboratory use of zebrafish (Danio rerio), (the 4th edition, Univ.of Oregon Press, Eugene, 2000).
Only if definition in addition, otherwise all scientific and technical terminologies used herein all have by the common identical implication of understanding of the those of ordinary skill of technical field under the present invention.Though similar or be equal to that method as herein described and material can be used for implementing or test the present invention, suitable method and material are described below.Mentioned all publications, patent application, patent and other list of references of this paper all by reference integral body incorporate this paper into.Occurring under the situation of contradiction, will explain that (comprising definition) is as the criterion with this.In addition, material, method and instance are merely illustrative and are not intended to restriction.
Embodiment
Describe the present invention with reference to following examples, these embodiment provide through the mode of explaining and are not to be intended to limit by any way the present invention.Use the specifically described technology of standard technique well known in the art or hereinafter.
Embodiment 1: the preparation of double-stranded RNA oligonucleotides
Synthetic and the purification of oligonucleotide
Can with the DsiRNA MOLECULE DESIGN be with RNA information in various sites (for example, RNA sequence as herein described in target sequence) interact.Use method as herein described with the synthetic DsiRNA molecule of chemical mode.Usually, use to the described solid phase oligonucleotide of 19 to 23mer siRNA synthetic method synthesize the DsiRNA construct (for example, referring to Usman etc., United States Patent (USP) the 5th, 804, No. 683; The 5th, 831, No. 071; The 5th, 998, No. 203; The 6th, 117, No. 657; The 6th, 353, No. 098; The 6th, 362, No. 323; The 6th, 437, No. 117; The 6th, 469, No. 158; Scaringe etc., United States Patent (USP) the 6th, 111, No. 086; The 6th, 008, No. 400; The 6th, 111, No. 086).
According to standard method synthesize the single rna chain and carry out the HPLC purification (Integrated DNA Technologies, Coralville, Iowa).For example, use the synthetic RNA oligonucleotide of solid phase phosphoramidite chemical agent, use standard technique to go protection and at NAP-5 post (Amersham Pharmacia Biotech; Piscataway; N.J.) go up desalination (Damha and Olgivie, 1993, Methods Mol Biol 20:81-114; Wincott etc., 1995, Nucleic Acids Res 23:2677-84).Use 15min grading linear gradient, at Amersham Source 15Q post (1.0cm * 25cm; Amersham Pharmacia Biotech, Piscataway N.J.) goes up use ion exchange HPLC (IE-HPLC) and comes the purification oligomer.This gradient was from 90: 10 buffer A: B becomes 52: 48 buffer A: B, and wherein buffer A is 100mM Tris (pH 8.5), and buffer B is 100mM Tris (pH 8.5), 1M NaCl.Monitoring sample under 260nm, and gather peak value corresponding to the full length rna oligonucleotide kind, compile desalination on the NAP-5 post, and lyophilizing.
Through capillary electrophoresis (CE), (Fullerton Calif.) goes up the purity of measuring every kind of oligomer for Beckman Coulter, Inc. at Beckman PACE 5000.CE internal diameter capillaceous is 100m and contains ssDNA 100R gel (Beckman-Coulter).Usually, the oligonucleotide injection capillary tube with about 0.6nmol carries out electrophoresis, and under 260nm, detects the UV absorbance in the electric field of 444V/cm.Buy degeneration Tris-borate-7M-carbamide electrophoretic buffer from Beckman-Coulter.Obtaining measuring purity through CE tests for being used for the following stated at least 90% oligoribonucleotide.Follow the scheme of manufacturer recommendation; Through Voyager DE.TM.Biospectometry Work Station (Applied Biosystems; Foster City, the characteristic of (MALDI-TOF) the mass spectroscopy chemical compound of substance assistant laser desorpted ionized flight time on Calif.).Obtain the relative molecular mass of all oligomers, usually the molecular mass of expectation 0.2% within.
The preparation of duplex
In the duplex buffer, be made up of 100mM potassium acetate, 30mM HEPES (pH 7.5) by this duplex buffer with (for example) 100mM concentration resuspending for single stranded RNA (ssRNA) oligomer.With equimolar amounts complementary sense strand and antisense strand are mixed, to produce the final solution of (for example) 50mM duplex.In RNA buffer (IDT), sample is heated to 100 ℃ and kept 5 minutes, and its cool to room temperature is for use.Store double-stranded RNA (dsRNA) oligomer down at-20 ℃.With single stranded RNA oligomer lyophilizing storage or be stored in-80 ℃ and do not contain in the water of nuclease.
Nomenclature
In order to keep consistency, in this explanation, adopted following nomenclature.Offer duplex title indication oligomer length and outstanding existence whether." 25/27 " is sense strand with 25 bases asymmetrical duplex with the antisense strand of 27 bases with 3 of 2 bases ' outstanding." 27/25 " is to have the sense strand of 27 bases and have the asymmetrical duplex of the antisense strand of 25 bases.
Cell culture and RNA transfection
Obtain the HeLa cell from ATCC, and 37 ℃ with 5%CO2 under, maintain be supplemented with 10% hyclone (HyClone) her Ge Shi culture medium (Dulbecco s modified Eagle medium) of Du Beikashi modified form (HyClone) in.Concerning the RNA transfection, use Lipofectamine TMRNAiMAX (Invitrogen) and follow the explanation of manufacturer is expressed as the DsiRNA transfection HeLa cell of 1nM or 0.1nM with ultimate density.Briefly, with 0.2 μ M or the Opti-MEM I (Invitrogen) of 0.02 μ M storing solution and 46.5 μ L and the Lipofectamine of 1 μ L of every kind of DsiRNA of 2.5 μ L TMRNAiMAX mixes.Gained 50 μ L mixture are added in each hole of 12 orifice plates, and at room temperature hatch 20min, to allow DsiRNA:Lipofectamine TMForm complex.Simultaneously, with trypsin treatment HeLa cell and with its with the ultimate density resuspending of 367 cells/μ L in culture medium.Finally, the cell suspending liquid of 450 μ L is added in each hole (final volume 500 μ L) and plate is positioned in the incubator cultivated 24 hours.
The assessment that suppresses
Through qRT-PCR measure target gene strike low, simultaneously to only comprising Lipofectamine TMThe HPRT of RNAiMAX (vehicle contrast) expresses the control treatment group or untreated fish group will be worth normalization.
The separation of RNA and analysis
With the PBS of 2mL once, and use RNeasy Mini Kit with cell washing TM(Qiagen) extract total RNA and in the final volume of 30 μ L, carry out elution.Follow the explanation of manufacturer, use Transcriptor 1st Strand cDNA KitTM (Roche) and total RNA of hexamer reverse transcription 1 μ g at random.CDNA (0.66 μ L) and the IQ Multiplex Powermix (Bio-Rad) of 5 μ L and the H of 3.33 μ L with a thirtieth gained 2The 3 μ M mixture of O and 1 μ L mix, and this mixture contains has specific primer and probe to people's gene HPRT-1 (registration number NM_000194) and KRAS target sequence.
Quantitative RT-PCR
The CFX96 real-time system that will have C1000 thermal cycler (Bio-Rad) is used for amplified reaction.The PCR condition is: 95 ℃, kept 3 minutes; Circulated 10 seconds down at 95 ℃ subsequently; 55 ℃ following 1 minute, 40 times the circulation.Each sample is carried out three retests.Relative HPRT mRNA level is directed against the horizontal normalization of KRAS mRNA, and compares with the mRNA level that in control sample of only handling or untreated samples, obtains with transfection reagent.Use Bio-Rad CFX Manager1.0 version software analytical data.
Embodiment 2:DsiRNA-sends the preparation and the use of peptide conjugate
Based on the synthetic oligonucleotide of the present invention of the chemical action of puting together of HyNic (6-hydrazino-nicotinamide) modified peptides and 4FB (4-formoxyl virtue amide) modified oligonucleotide-send peptide conjugate.Also can use other peptide synthetic method known in the art and put together program.
On N end or C end, use respectively 6-Boc-HyNic or FMOC-Lys-(OH of ε-6-BocHyNic) with HyNic partly and on peptide.Use TFA/ acetone/tri isopropyl silane (TIS) (92.5/2.5/2.5/2.5) 2 hours, and accomplished cracking from resin.The existence of acetone is formed on has the hydrazone of protecting the hydrazine part, forms any trifluoroacetamide thereby stop in position from the reaction of TFA and strong nucleophilic hydrazine.Analyze rough peptide by HPLC and ES-MS.Use gradient method, by the RP-HPLC separated product.Concerning the Peg12 peptide, between synthesis stage, directly add the Polyethylene Glycol synthon in solid-phase peptide.In some instances, also use Polyethylene Glycol oligonucleotide synthon, other Polyethylene Glycol sept is added into the oligonucleotide end.
Amido modified oligonucleotide is converted into 5 '-the 4FB-oligonucleotide.When the HyNic-of 2-5 molar excess peptide, carry out being connected of oligonucleotide that HyNic-peptide and 4FB modify, and produce conjugate productive rate usually greater than 80%.Through including aniline in, catalysis hydrazone key forms and reaction power is increased to 10-100 doubly, causes usually puting together productive rate greater than 95%.The best power (formation of hydrazone key) of puting together reaches at pH 4.5-5.0.Yet reaction can also be carried out under higher pH, although speed is slower.Confirm every kind of optimum pH that puts together by rule of thumb, also will consider the dissolubility of different peptide sequences.Puting together degree can be through the spectrophotometry technical monitoring.The formation of two aryl hydrazone keys not only had been used to the progress of following the trail of conjugation reaction but also being used for quantitative conjugation reaction, wherein use known molar extinction coefficient (29,000354nm).Use diafiltration to remove excessive peptide, thereby produce oligonucleotide-peptide conjugate.In order to produce the peptide of HyNic quencher, make the reaction of HyNic-peptide and 2-sulfo group benzaldehyde, so that the HyNic reactive moieties inactivation on the peptide.
Acellular restriction analysis
(Genlantis #T52002) is hatched 2 hours to the DsiRNA (ultimate density is 5 μ M) that under 37 ℃, DsiRNA or peptide is puted together, and uses stop buffer that reaction is stopped with recombined human dicer enzymatic mixture.With this final solution and gel loading buffer (Bio-Rad, #161-0767) mixing.Cracked dsRNA of natural polypropylene acrylamide gel electrophoretic analysis Dicer and complete DsiRNA through 18%.Use Bio-Rad VersaDocTM imaging system (model 4000MP) to obtain gel images.
Serum stability is analyzed
Under 37 ℃, the DsiRNA (ultimate density is 2 μ M) that DsiRNA or peptide are puted together was hatched 2 hours in 90% (v/v) mice serum (Sigma#M5905).In different time point (0,2,4,8,1,10 and 25 hour), 10 μ L samples are mixed with 2 μ L H2O and 3 μ L gel loading buffers (Bio-Rad#161-0767), and be about to said sample moment in ethanol the dry ice bath neutrality and freeze.Make sample go up electrophoresis at 18% natural polypropylene acrylamide gel (Bio-Rad#161-1216).Use Bio-Rad VersaDocTM imaging system (Bio-Rad model#4000MP) quantitative to the siRNA band of analyzing.Through drawing the dsRNA band strength over time, calculate the half-life of single dsRNA in 90% serum.
HPRT1 targeting DsiRNA and KRAS targeting DsiRNA
As described herein, synthetic exemplary DsiRNA to HPRT1 and KRAS target gene, wherein DsiRNA have oligonucleotide sequence, 2 '-end modified shown in O-methyl and Fig. 2.
Put together peptide
In Fig. 3, listed and be used for or can be used in the exemplary delivery peptide of puting together with DsiRNA of the present invention, Fig. 3 also indicates and every kind of single relevant combination target of peptide of sending.
Through observing and successfully puting together relevant size increase (with the consequent electrophoretic mobility that is stagnated), confirm that the success of various peptides-DsiRNA conjugate is synthetic.Shown in Fig. 4 to 6, HPRT1 targeting DsiRNA and KRAS targeting DsiRNA all successfully put together with several peptides, thereby form following conjugate: K1459-SEQ ID NO:118; K1459-Peg12-SEQ ID NO:118; K1379-SEQ ID NO:118; K1379-Peg12-SEQ ID NO:118 (Fig. 4); H1460-SEQ ID NO:118; H1460-Peg12-SEQ ID NO:118 (Fig. 5); K1379-SEQ ID NO:149; K1379-SEQ ID NO:154; K1379-SEQ ID NO:155; And K1379-SEQ ID NO:156 (Fig. 6).
Also successfully synthetic DsiRNA-peptide conjugate with cleavable connexon part (for example, disulphide group).Specifically, Fig. 7 shows the conjugate of the stable and cleavable that has generated SEQ ID NO:118 with KRAS targeting DsiRNAK1379 and SEQ ID NO:120.
DsiRNA also puts together with cyclic peptide.As shown in Figure 8, KRAS targeting DsiRNA (K1459) successfully puts together with cyclic peptide SEQ ID NO:151.
For the Dicer cracking of evaluate exemplary peptide-DsiRNA conjugate, as stated conjugate K1096-SEQ ID NO:120 conjugate is carried out acellular enzyme action and measure.As shown in Figure 9, confirm that this conjugate is by the Dicer cracking.
Embodiment 3: the dsRNA-of transfection sends peptide conjugate and reduces the cell gene expression dose that hits
Cell culture and RNA transfection
Obtain HeLa cell and HepG2 cell from ATCC, and under 37 ℃ and 5%CO2, maintain in her the Ge Shi culture medium (HyClone) of Du Beikashi modified form that is supplemented with 10% hyclone (HyClone).DsRNA and dsRNA are sent peptide put together the transfection, at Lipofectamine TMRNAiMAX (Invitrogen) exists down, does not put together or put together the DsiRNA transfection HeLa cell with what indicate ultimate density (for example, 1nM or 0.1nM).In some instance, do not put together DsiRNA also as positive control.In some instance, 0.2 μ M or the 0.02 μ M storing solution of every kind of DsiRNA of 2.5 μ L mixed with the Opti-MEM I (Invitrogen) of 47.5 μ L.To Lipofectamine TMContrast is with 0.2 μ M or the Opti-MEM I (Invitrogen) of 0.02 μ M storing solution and 46.5 μ L and the Lipofectamine of 1 μ L of every kind of DsiRNA of 2.5 μ L TMRNAiMAX mixes.Gained 50 μ L mixture are added in each hole of 12 orifice plates, and at room temperature gained 50 μ L mixture were cultivated 20 minutes, form to allow DsiRNA:LipofectamineTM RNAiMAX complex.Simultaneously, use trypsin treatment HeLa cell or HepG2 cell and be that about 367 cells/μ L resuspending is in culture medium with ultimate density with it.Finally, the cell suspending liquid of 450 μ L is added in each hole (final volume 500 μ L) and plate is positioned in the incubator cultivated 24 hours.Concerning dose response research, the concentration of transfection DsiRNA is changed to 1nM from initial 1pM.Concerning dose response research (relating to the DsiRNA-peptide conjugate of using to cell under the vectorial situation of transfection not existing), the concentration of DsiRNA that is used and DsiRNA-peptide conjugate changes to about 5mM from about 5nM.Can also carry out time course research, wherein study about 4 hours to about 72 hours incubation time.
The assessment that suppresses
Through qRT-PCR measure target gene strike low, simultaneously to only comprising Lipofectamine TMThe HPRT of RNAiMAX (vehicle contrast) expresses the control treatment group or untreated fish group will be worth normalization.
RNA separates and analyzes
With the PBS of 2mL once, and use RNeasy Mini Kit with cell washing TM(Qiagen) extract total RNA and in the final volume of 30 μ L, carry out elution.Follow the explanation of manufacturer, use Transcriptor 1st Strand cDNA Kit TM(Roche) and at random total RNA of hexamer reverse transcription 1 μ g.CDNA (0.66 μ L) and the IQ Multiplex Powermix (Bio-Rad) of 5 μ L and the H of 3.33 μ L with a thirtieth gained 2The 3 μ M mixture of O and 1 μ L mix, and this mixture contains has specific primer and probe to people's gene HPRT-1 (registration number NM_000194) and KRAS target sequence.
Quantitative RT-PCR
The CFX96 real-time system that will have C1000 thermal cycler (Bio-Rad) is used for amplified reaction.The PCR condition is: 95 ℃, kept 3 minutes; Circulated 10 seconds down at 95 ℃ subsequently; 55 ℃ following 1 minute, 40 times the circulation.Each sample is carried out twice retest (data of the repeated experiments of wherein carrying out to every kind of reagent place are shown in Fig. 2 to 19).Relative HPRT mRNA level is directed against the horizontal normalization of KRAS mRNA, and compares with the mRNA level that in control sample of only handling or untreated samples, obtains with transfection reagent.Use Bio-Rad CFX Manager1.0 version software analytical data.Expression data is rendered as in the comparison of handling the expression that the expression do not put together under the dsRNA and dsRNA-send peptide conjugate.
When using, check that at first the DsiRNA-peptide conjugate suppresses the ability of said target mrna level in the cell through transfection.As shown in Figure 10, when being applied to the HeLa cell through transfection under with the concentration of 0.1nM and Geng Gao, observing KRAS targeting DsiRNA K1096-SEQ ID NO:120 the said target mrna level is suppressed at least 80%.Observe to the peptide-being seen target gene inhibition of DsiRNA conjugate level and be similar to the free being seen target gene inhibition of K1096DsiRNA level.Therefore, when being applied to the HeLa cell, observe effective inhibitor that peptide-DsiRNA conjugate is the target rna level through transfection.
The serum stability that embodiment 4:DsiRNA-peptide conjugate confirms
The serum stability of assessment DsiRNA reagent as described herein.As shown in Figure 11; And have other identical modification pattern but do not have another DsiRNA of SEQ ID NO:120 peptide to compare, DsiRNA K1096 and SEQ ID NO:120 peptide put together the half-life significant prolongation that can cause puting together agent (K1096-SEQ ID NO:120 (half-life is 13.3 hours)).
Embodiment 5: the dsRNA-peptide conjugate of being used when not having transfection vehicle (for example, dsRNA sends peptide conjugate) reduces target gene expression level in the cell
For the DsiRNA-peptide conjugate of assessing example promotes this type of to put together the ability that agent is delivered to target cell having no under the vectorial situation of transfection, in the concentration range of 20nM to 2 μ M, use a series of DsiRNA-peptide conjugates to the HeLa cell.As shown in Figure 12, and compared by the DsiRNA of transfection, the concentration of the rising of DsiRNA-peptide conjugate is that to reach remarkable inhibition said target mrna level needed.Yet the conjugate of two tests (K1096-Peg12-SEQ ID NO:118 and K1096-SEQ ID NO:120) shows with the dose response mode, and wherein two kinds under 2 μ M concentration are puted together the target gene of observing significant level the molecule and suppressed.In fact, K1096-SEQ ID NO:120 conjugate has shown in the down beat all and significant said target mrna level reduction of all test concentrations (20nM, 200nM and 2 μ M).
Subsequently, when assessment is applied to the HepG2 cell with the concentration of 5mM when not having the vectorial situation of transfection under, the ability of DsiRNA-peptide conjugate K1379-SEQ ID NO:118 and K1379-SEQ ID NO:120 inhibition KRAS target gene.As shown in Figure 13, clearly,, observe the said target mrna level and reduced more than 90% not existing under the vectorial situation of transfection concentration with 5mM to be applied to two kinds of conjugates of HepG2 cell.This type of result is similar to 1nM K1379DsiRNA is applied to the observed result of HepG2 cell separately.Beat allly be that the quencher peptide (the SEQ ID NO:120 of the SEQ ID NO:118 of quencher or quencher) that will have a free DsiRNA K1379 is included in and caused any said target mrna to suppress active completely losing in the mixture that is applied to the HepG2 cell.
In order to check whether exemplary DsiRNA-peptide conjugate is than the corresponding free horizontal inhibitor of the more effective said target mrna of DsiRNA under the situation that does not have delivery vehicle; Obtain dose response curve; Free K1379DsiRNA of this curve ratio and K1379-SEQ ID NO:118 conjugate, and relatively more free K1379DsiRNA and K1379-SEQ ID NO:120 conjugate.As shown in Figure 14, at all IC 50In the information concentration, the K1379-peptide conjugate is obviously good than corresponding free K1379DsiRNA performance.In fact, compare, do not have IC measured under the vectorial situation of transfection the DsiRNA-peptide conjugate with corresponding D siRNA-peptide conjugate 50Value is to the free measured IC of DsiRNA 50Not 2 to 3 times (therefore, so ineffective) of value.
Embodiment 6: the preparation of sending peptide-dsRNA conjugate and/or targeting peptide-dsRNA conjugate in addition
Preferred target DsiRNA reagent in addition is selected from the prescreen crowd of DsiRNA.The design of DsiRNA can randomly relate to uses the predictability scoring algorithm, and its activity/effect to the expectation of several possibilities DsiRNA of leap sequence area is carried out the computer simulation assessment.
Through in this paper said method any, with dsRNA of the present invention with send peptide or the targeting peptide is puted together.Use the maleimide chemical action, make about 20mg have 5 ' amino DsiRNA (～1 μ mol) and 3 to 5 moles excessive reactive polypeptide (Moschos etc., Bioconjug Chem.2007 with terminal cysteine sulfydryl; 18 (5): 1450-9; Nishina etc., Mol Ther.2008; 16 (4): 734-40).Through diafiltration peptide-RNA conjugate is carried out purification to remove excessive peptide, make peptide-RNA conjugate desalination and as the freeze-dried powder supply.Use deconvolution to measure the purity of end product through analytical anion exchange HPLC and electrojet mass spectrum.
Embodiment 7: use dsRNA-targeting peptide conjugate to reduce target gene expression in the cell in addition
Cell culture and RNA transfection
From ATCC obtain HeLa, Hep3B, HepG2, HT29, LS174T and Neuro2a and under 37 ℃ at 5% CO 2Following, and maintain in the basal medium of the recommendation with 10% heat-inactivated FBS.Concerning dsRNA and the transfection of dsRNA-targeting peptide conjugate, use as be marked as ultimate density to be 1nM or 0.1nM Lipofectamine TMThe DsiRNA transfectional cell of not puting together or puting together of RNAiMAX (Invitrogen).DsiRNA is as positive control.Briefly, 0.2 μ M or the 0.02 μ M storing solution with every kind of DsiRNA of 2.5 μ L mixes with the Opti-MEM I (Invitrogen) of 47.5 μ L.To Lipofectamine TMContrast is with 0.2 μ M or the Opti-MEM I (Invitrogen) of 0.02 μ M storing solution and 46.5 μ L and the Lipofectamine of 1 μ L of every kind of DsiRNA of 2.5 μ L TMRNAiMAX mixes.Gained 50 μ L mixture are added in each hole of 12 orifice plates, and at room temperature hatch 20min, to allow DsiRNA:Lipofectamine TMThe RNAiMAX complex forms.Simultaneously, with the trypsin treatment cell and with its with the ultimate density resuspending of 367 cells/μ L in culture medium.Finally, the cell suspending liquid of 450 μ L is added in each hole (final volume 500 μ L) and plate is positioned in the incubator cultivated 24 hours.Concerning dose response research, the concentration of DsiRNA is changed to 1nM from initial 1pM.Concerning time course research, study about 4 hours to about 72 hours incubation time.
The assessment that suppresses
Through qRT-PCR measure target gene strike low, simultaneously to only comprising Lipofectamine TMThe HPRT of RNAiMAX (vehicle contrast) expresses the control treatment group or untreated fish group will be worth normalization.
RNA separates and analyzes
With the PBS of 2mL once, and use RNeasy Mini Kit with cell washing TM(Qiagen) extract total RNA and in the final volume of 30 μ L, carry out elution.Follow the explanation of manufacturer, use Transcriptor 1st Strand cDNA KitTM (Roche) and total RNA of hexamer reverse transcription 1 μ g at random.(0.66 μ L) and the IQ Multiplex Powermix (Bio-Rad) of 5 μ L and the H2O of 3.33 μ L and the 3 μ M mixture of 1 μ L mix among the cDNA with a thirtieth gained, and this mixture contains has specific primer and probe to people's gene HPRT-1 (registration number NM_000194) and KRAS target sequence.
Quantitative RT-PCR
The CFX96 real-time system that will have C1000 thermal cycler (Bio-Rad) is used for amplified reaction.The PCR condition is: 95 ℃, kept 3 minutes; Circulated 10 seconds down at 95 ℃ subsequently; 55 ℃ following 1 minute, 40 times the circulation.Each sample is carried out three retests.Relative HPRT mRNA level is directed against the horizontal normalization of KRAS mRNA, and compares with the mRNA level that in control sample of only handling or untreated samples, obtains with transfection reagent.Use Bio-Rad CFX Manager1.0 version software analytical data.Expression data is rendered as the comparison in the expression of handling the expression do not put together under the dsRNA and dsRNA-targeting peptide conjugate.
Embodiment 8: use dsRNA-to send peptide conjugate to reduce target gene expression in the animal
For delivery efficiency and the follow-up function of assessing dsRNA, use peptide and dsRNA-to send peptide conjugate, subcutaneous (s.c.) tumor model (Judge etc., J Clin Invest.2009; 119 (3): 661-73).Through with 3 * 10 among the 50 μ LPBS 6Individual cell skin injected is set up the Hep3B tumor in female SCID/beige mice to left rear side.Along with tumor becomes obviously, mice is divided into many processed group at random at postvaccinal 10-17 days.Through calculating based on the mg dsRNA/kg body weight according to individual animals weight, dsRNA, peptide and dsRNA send the preparation or the contrast of PBS vehicle of peptide conjugate and use through injecting via outside tail venous standard intravenous (i.v).Use digital caliper aspect two dimension (width * length), to measure tumor with the assessment tumor growth.Gross tumor volume utilizes equality x*y*y/2 to calculate (wherein x=maximum gauge, and y=minimum diameter), and representes with cell mean ± SD.Also remove tumor tissues, and confirm that clpp gene is low from the animal of different disposal group.Gross tumor volume, survival and rna expression data are rendered as the comparison between the processing of the non-dsRNA of puting together and the processing that dsRNA-sends peptide conjugate.
Embodiment 9: use dsRNA-targeting peptide conjugate to reduce target gene expression in the cell
For targeting efficient and the follow-up function of assessing dsRNA, use tumor model (Judge etc., J Clin Invest.2009 in peptide and dsRNA-targeting peptide conjugate, the liver; 119 (3): 661-73).Through direct injection into liver Hep3B or Neuro2a tumor cell, in mice, set up liver neoplasm.Female SCID/beige mice and male A/J mice are used as the host of Hep3B and Neuro2a tumor respectively.Mice is maintained under the gas anesthesia state, below breastbone, do single 1.5cm otch, and the left side lobe of the liver is taken out across center line.Use Hamilton syringe and 30 gage needles will be suspended in 1 * 10 among the 25 μ L PBS 6Hep3B cell or 1 * 10 5The Neuro2a cell slowly is injected in the said lobe of the liver with low-angle.Then swab is put on puncture wound with hemostasis before sewing up.Before mice is put back to conventional residence, mice is recovered from narcotism in aseptic cage, and pay close attention to mice and reach 2-4 hour.After tumor is implanted eight to 11 days; Through calculating based on mg dsRNA/kg body weight according to individual animals weight; Mice is divided into dsRNA, peptide and dsRNA peptide conjugate preparation or PBS vehicle control treatment group at random, and these reagent are through using via outside tail venous standard intravenous (i.v.) injection.In whole research process, the monitoring body weight is as the indicant of tumor load in the development and treatment toleration.Concerning efficacy study, fixed people's terminal point is confirmed as the substitute of existence.Based on clinical symptoms, lose weight and the combination of abdominal distention is assessed the date of the euthanasia that causes by tumor load with definition.From the animal of different disposal group, remove tumor tissues, and confirm that clpp gene is low.
Also through with fluorescence labels labelling peptide and/or dsRNA and use live animal imaging system (Xenogen or BioRad) to carry out the biological research that distributes; Functional (the Eguchi etc. of the tumor cell targeting of test peptides, dsRNA and dsRNA-peptide conjugate; Nat Biotechnol.2009 May 17, [delivering preceding publication]).Make in this way, and, independent peptide and dsRNA-peptide conjugate are confirmed peptide and the bonded ability of tumor cell through comparing with free (that is, unconjugated) dsRNA.
On the contrary, the unconjugated dsRNA as the contrast in this research can not combine with tumor surface with the degree identical with the dsRNA that has puted together.Effect terminal point, rna expression and bio distribution data are rendered as with the processing of unconjugated dsRNA and with the comparison between the processing of dsRNA-targeting peptide conjugate.
Embodiment 10: use other cell culture model to assess the downward modulation of KRAS gene expression
Various terminal points have been used for cell culture model, to observe the Ras mediation after handling with anti-Ras reagent.The phenotype terminal point comprises the inhibition of cell proliferation, the inhibition of rna expression and the reduction of Ras protein expression.Because the sudden change of KRAS oncogene is contacted directly with the propagation increase of some tumor cell, so the propagation terminal point of measuring to cell culture preferably is used as elementary screening.There are the some kinds of methods that can measure this terminal point.After handling cell with the conjugate of DsiRNA of the present invention and peptide, make cell growth (common 5 days), can measure after this in cell viability, the cell DNA [ 3H] the incorporating into and/or cell density of thymidine.Can use commercially available fluorescence nucleic acid stain (such as,
Figure BDA0000133674250002501
or
Figure BDA0000133674250002502
) carry out the analysis of cell density with 96 well format.As terminal point secondary, that confirm, can use the peptide-mediated reduction of DsiRNA-of the specific ELISA assessment of KRa KRas protein expression level.
Embodiment 11: assess the effect of anti-KRAS DsiRNA in the mouse model of KRAS mistake regulation and control
Can be in mouse model (comprising for example xenograft and above-mentioned other animal model), further test is selected from the anti-KRAS DsiRNA-peptide conjugate in the analyzed in vitro.In one embodiment, through injecting in the hydrodynamic tail vein, use DsiRNA-peptide reagent of the present invention to the mice of (for example, improving) KRAS level with wrong regulation and control.DsiRNA to every group of (the specific DsiRNA reagent based on through test a divides into groups) 3-4 injected in mice 50 μ g or 200 μ g.Use the level of RT-qPCR assessment KRAS RNA.In addition or alternatively; The level that can use art-recognized method assessment KRas (for example; KRas protein level and/or cancerous cell/tumor forms, grows or diffusion); Or monitoring (randomly, as the representative of measuring KRAS transcript or KRas protein level) is regulated and control relevant phenotype (for example, tumor formation, growth, transfer etc.) with the mistake of KRAS.Then, can also combined standard chemistry therapy test the active DsiRNA-peptide conjugate in this type of animal model.
Embodiment 12: diagnostic uses
DsRNA-peptide molecule of the present invention can be used in the multiple Diagnosis Application, differentiates the molecule target (for example, RNA) such as being used in the multiple application (for example, in clinical, industrial, environment, agricultural and/or the research background).This type of diagnosis of dsRNA-peptide molecule utilizes and to relate to the RNAi system that rebuilds that utilizes, and for example, uses cell lysate or through partially purified cell lysate.DsRNA-peptide molecule of the present invention can be used as diagnostic tool with genetic drift and sudden change in the check diseased cells.Substantial connection between the active structure with target RNA of dsRNA-peptide makes the sudden change in any district that can detect target molecule, base pairing and the three dimensional structure of this sudden change change target RNA.Through using the dsRNA-peptide molecule described in a plurality of the present invention, can change the external and cell of the RNA nucleotide very important and map with organizing inner structure and function.Can be used for the developing effect of inhibition of gene expression and definite specific gene product with DsiRNA molecule cracking target RNA in disease that is associated with particular target or disease.Mode after this manner can be confirmed as other hereditary target the important amboceptor of disease.These experiments will draw through the probability that combination treatment (for example, with different genes as a plurality of DsiRNA molecules of target, with the link coupled DsiRNA molecule of known small molecules inhibitor or with the intermittent therapy of the combination of DsiRNA molecule and/or other chemistry or biomolecule) is provided PD will better be treated.The external purposes of other of DsiRNA molecule of the present invention is well-known in the art, and comprises the existence of detection and disease or the conditions associated RNA that is associated.This type of RNA detects through measuring existing of pyrolysis product after handling with DsiRNA in use standard method (for example, (FRET) shifted in the fluorescence resonance radiation).
In a particular embodiment, only the DsiRNA molecule of the target KRAS RNA of cracking wild type or saltant or polymorphism is used for measuring.The one DsiRNA molecule (promptly; Those DsiRNA molecules of only cracking wildtype target KRAS RNA) be used for identifying the wild type KRAS RNA that is present in sample; And the 2nd DsiRNA molecule (that is the DsiRNA molecule of only cracking saltant or polymorphism target RNA) is used for identifying the saltant or the polymorphism KRAS RNA of sample.With the synthetic substrate of two kinds of DsiRNA molecule cracking wild types and saltant or polymorphism KRAS RNA as the reaction pair photograph, with explanation in " non-target " KRAS RNA kind lytic response with the relative DsiRNA effect not.The pyrolysis product that is obtained by synthetic substrate also is used for producing the dimension mark thing that supplies analytic sample crowd's wild type and saltant KRAS RNA.Therefore, each analysis needs two kinds of DsiRNA molecules, two kinds of substrates and a kind of unknown sample, thereby is combined into six reactions.Utilize the existence of ribonuclease protecting test determination pyrolysis product, so that can in a swimming lane of PAAG, analyze total length and the crack fragment of each KRAS RNA.Be not absolute requirement quantitatively these results to understand supposition risk to the related phenotype variation of KRAS in the expression of saltant or polymorphism KRAS RNA and the target cell.The expression that protein relates to the KRAS mRNA in the evolution of phenotype (that is, disease relevant/related) is enough to confirm risk.If use the probe that two kinds of transcripies is had suitable specific activity, then the qualitative comparison of KRAS rna level is just enough, and reduces the cost of initial diagnostic.No matter be relatively qualitative or Quantitative Comparison KRAS rna level, the ratio of higher saltant or polymorphism and wild type is all interrelated with higher risk.
Mentioned all patents and publication are all indicated the technical merit with one of ordinary skill in the art of the present invention in this description.All lists of references of being quoted in the disclosure are all incorporated into way of reference, and the degree of quoting is as each list of references is incorporated herein with way of reference independently in full.
Those skilled in the art will be easy to understand, and the present invention can be suitable for reaching mentioned purpose preferably and obtain mentioned and wherein inherent result and benefit.Method and composition as herein described like present representative preferred embodiment is exemplary, and is not intended to limit scope of the present invention.Those skilled in the art can expect the change and other purposes of these method and compositions, and these all are encompassed in the spirit of the present invention that scope defined by claim.
It will be evident to those skilled in the art that under the situation that does not deviate from scope of the present invention and spirit and can advance different rows replacement and modification invention disclosed herein.Therefore, these other embodiments are also within the scope of the present invention and following claims.The present invention instructs those skilled in the art to be directed against and produces to have and is used for various combinations and/or the replacement that the active nucleic acid construct of the active improvement of mediate rna i is tested chemical modification as herein described.This kind improves the active improved activation that possibly comprise the cell effect that improves stability, improvement bioavailability and/or mediate rna i.Therefore; Particular as herein described is also nonrestrictive, and one of ordinary skill in the art possibly be easy to recognize: can not have to have the particular combination of improving the modification as herein described of the active DsiRNA molecular testing of RNAi to identification under the situation of improper experiment.
The present invention that illustrative is described among this paper should implement under the situation that does not have not disclosed especially any or multiple key element, one or more restrictions among this paper.Therefore, for example, in each situation of this paper, term " comprises ", " basically by ... form " with " by ... form " in any one can use in other two terms any one alternative.These terms that adopted are description rather than restriction as term with statement; And be not intended to use these terms and explain shown in the eliminating and any equivalent of described characteristic or its part; But it should be understood that the various modifications in desired scope of the present invention all are possible.Therefore; Should be appreciated that; Although disclose the present invention particularly through embodiment preferred; But those of skill in the art can adopt optional characteristic, modification and the change of the disclosed idea of this paper, and think these modifications and change describe and scope that accompanying claims is defined in.
In addition; Describe according to Ma Kushi group (Markush group) or another substituting grouping characteristic of the present invention or aspect the time; Those of skill in the art it should be understood that equally can describe the present invention according to the arbitrary indivedual members or the membership group of this Ma Kushi group or other group in view of the above.
Only if indicate in addition among this paper or the obvious contradiction of context, otherwise the term " " that (in the context of especially following claim) uses in describing context of the present invention, " a kind of " and " said " and similar refer to word and should be interpreted as encompasses singular and plural number.Unless otherwise indicated, otherwise term " comprises ", " having ", " comprising " and " containing " should be interpreted as open-ended term (that is the meaning of expression " including but not limited to ").The enumerating of the scope of this paper intermediate value only is intended to as each method of writing a Chinese character in simplified form of value separately of mentioning separately in this scope, only if indicate in addition among this paper, otherwise each independent value all incorporates in this description, as in this article it being enumerated separately.Only if indicate in addition among this paper or the obvious contradiction of context, otherwise all methods described herein can be undertaken by any suitable order.Only if in addition requirement, otherwise the use of any and all embodiment of providing among this paper or exemplary language (for example, " as ") all only is intended to better set forth the present invention, rather than the scope of the present invention or claim constituted limit.Not having language should be interpreted as any non-element that requires of indication in the description is absolutely necessary for enforcement of the present invention.
Embodiment of the present invention have been described in this article, comprising the best mode that is used for embodiment of the present invention for the inventor knew.After reading above-mentioned explanation, the variation of those embodiments will become obvious to those of ordinary skill in the art.
The inventor expects that those of skill in the art take the circumstances into consideration to adopt this type of to change, and the inventor hopes to come embodiment of the present invention with the mode beyond the specifically described mode of this paper.Therefore, under the situation that applicable law allows, present invention resides in all modifications and the equivalent of being stated in the appended claims of the present invention.In addition, any combination of the above-mentioned key element in of the present invention might the variation is contained by the present invention, only if statement or obviously contradictory with context is arranged among this paper in addition.Those of skill in the art only use normal experiment just will recognize the many equivalents that maybe can confirm specific embodiments of the present invention described herein.Intention is encompassed in these equivalents in the above claim.

Claims (87)

1. an isolating double stranded RNA (dsRNA) compositions; It comprises and has 5 ' first oligonucleotide chain of end and 3 ' end and second oligonucleotide chain with 5 ' end and 3 ' hold; The length of wherein said first chain and said second chain is at least 16 and 50 nucleotide at the most, and wherein said peptide is puted together with said dsRNA and wherein said dsRNA-peptide conjugate combines with target.
2. an isolating double stranded RNA (dsRNA) compositions; It comprises and has 5 ' first oligonucleotide chain of end and 3 ' end and second oligonucleotide chain with 5 ' end and 3 ' hold; The length of wherein said first chain and said second chain is at least 16 and 50 nucleotide at the most; Wherein said peptide and said dsRNA put together, and wherein said dsRNA-peptide conjugate is by the target cell internalization.
3. according to claim 1 or claim 2 composition isolated, the length of wherein said first chain and said second chain is at least 25 and 35 nucleotide, at least 19 and 35 nucleotide, at least 19 and 24 nucleotide, at least 25 and 30 nucleotide, at least 26 and 30 nucleotide or at least 21 and 23 nucleotide at the most at the most at the most at the most at the most at the most.
4. according to claim 1 or claim 2 composition isolated, wherein said second chain comprises outstanding at 3 ' end.
5. according to claim 1 or claim 2 composition isolated, wherein said first chain comprises outstanding at 3 ' end.
6. according to claim 1 or claim 2 composition isolated, at least one in wherein said second chain and said first chain comprises outstanding at 3 ' end.
7. composition isolated as claimed in claim 6, the said 3 ' outstanding said nucleotide of wherein said first chain and/or second chain comprises the nucleotide through modifying.
8. composition isolated as claimed in claim 6, wherein said 3 ' outstanding length is 1 to 5 nucleotide.
9. according to claim 1 or claim 2 composition isolated, each the bar chain in wherein said first chain and said second chain is made up of the nucleotide residue of similar number.
10. composition isolated as claimed in claim 9, it is right that last residue of last residue of the said 5 ' end of wherein said first chain and the said 3 ' end of said second chain forms base mismatch.
11. composition isolated as claimed in claim 9, it is right that said last residue of said last residue of the said 3 ' end of wherein said first chain and the said 5 ' end of said second chain forms base mismatch.
12. composition isolated as claimed in claim 9, it is right that said last residue of said last residue of the said 5 ' end of wherein said first chain and the said 3 ' end of penult residue and said second chain and penult residue form two base mismatch.
13. composition isolated as claimed in claim 9, it is right that said last residue of said last residue of the said 3 ' end of wherein said first chain and the said 5 ' end of penult residue and said second chain and penult residue form two base mismatch.
14. composition isolated according to claim 1 or claim 2, wherein said peptide comprise 6-100 aminoacid.
15. composition isolated according to claim 1 or claim 2, wherein said peptide comprise 10-50 aminoacid.
16. composition isolated according to claim 1 or claim 2, wherein said peptide comprise 15-30 aminoacid.
17. require 1 or 2 described composition isolated like profit, wherein said peptide comprises 10 aminoacid.
18. composition isolated according to claim 1 or claim 2, wherein said dsRNA-peptide conjugate and receptors bind.
19. composition isolated according to claim 1 or claim 2, wherein said dsRNA-peptide conjugate combines with at least one member of said ldl receptor family.
20. composition isolated according to claim 1 or claim 2, wherein said peptide are the PAR part.
21. composition isolated according to claim 1 or claim 2, wherein said peptide are the PAR1 part.
22. composition isolated according to claim 1 or claim 2, wherein said peptide are the somatomedin parts.
23. composition isolated according to claim 1 or claim 2, wherein said peptide are insulin or insulin like growth factor part.
24. composition isolated according to claim 1 or claim 2, wherein said peptide are the IGF-1 parts.
25. composition isolated according to claim 1 or claim 2, wherein said peptide are the hormone parts.
26. composition isolated according to claim 1 or claim 2, wherein said peptide are the PTH parts.
27. composition isolated according to claim 1 or claim 2, wherein said peptide are the PTH-1 parts.
28. composition isolated according to claim 1 or claim 2, wherein said dsRNA-peptide conjugate combines with receptor binding protein.
29. composition isolated is according to claim 1 or claim 2 wherein puted together said peptide and said dsRNA with stable connexon.
30. composition isolated as claimed in claim 29, wherein said stable connexon comprises the homotype bi-functional cross-linking agent.
31. composition isolated as claimed in claim 29, wherein said stable connexon comprises special-shaped bi-functional cross-linking agent.
32. composition isolated as claimed in claim 29, wherein said stable connexon comprises three functional cross-link agents.
33. composition isolated is according to claim 1 or claim 2 wherein puted together said peptide and said dsRNA with the connexon of cleavable.
34. composition isolated as claimed in claim 33, the connexon of wherein said cleavable comprises the disulphide connexon.
35. composition isolated is according to claim 1 or claim 2 wherein puted together said peptide and said dsRNA with the carbon connexon.
36. composition isolated as claimed in claim 35, wherein said carbon connexon comprise 18 carbon at the most.
37. composition isolated as claimed in claim 35, wherein said carbon connexon comprises 6 carbon.
38. composition isolated is according to claim 1 or claim 2 wherein puted together said peptide and said dsRNA under the situation of no connexon.
39. composition isolated is according to claim 1 or claim 2 wherein puted together the said 3 ' end of said first chain of said peptide and said dsRNA.
40. composition isolated according to claim 1 or claim 2, the said 3 ' end of said second chain of wherein said peptide and said dsRNA is puted together.
41. composition isolated is according to claim 1 or claim 2 wherein puted together the said 5 ' end of said first chain of said peptide and said dsRNA.
42. composition isolated is according to claim 1 or claim 2 wherein puted together the said 5 ' end of said second chain of said peptide and said dsRNA.
43. composition isolated according to claim 1 or claim 2, wherein with said second chain of the said 5 ' end of said first chain of said peptide and said dsRNA and said dsRNA said 5 ' hold and put together.
44. composition isolated according to claim 1 or claim 2, wherein with said second chain of the said 5 ' end of said first chain of said peptide and said dsRNA and said dsRNA said 3 ' hold and put together.
45. composition isolated according to claim 1 or claim 2, wherein with said second chain of the said 3 ' end of said first chain of said peptide and said dsRNA and said dsRNA said 3 ' hold and put together.
46. composition isolated according to claim 1 or claim 2, wherein with said second chain of the said 3 ' end of said first chain of said peptide and said dsRNA and said dsRNA said 5 ' hold and put together.
47. composition isolated is according to claim 1 or claim 2 wherein puted together the said first chain inside of at least one peptide and said dsRNA.
48. composition isolated is according to claim 1 or claim 2 wherein puted together the said second chain inside of at least one peptide and said dsRNA.
49. composition isolated is according to claim 1 or claim 2 wherein puted together the said first chain inside of at least one peptide and said dsRNA and wherein the said second chain inside of at least one peptide and said dsRNA is puted together.
50. composition isolated is according to claim 1 or claim 2 wherein puted together two peptides and said dsRNA at least.
51. composition isolated as claimed in claim 50, wherein said at least two peptides are identical.
52. composition isolated as claimed in claim 50, wherein said at least two peptides are inequality.
53. composition isolated according to claim 1 or claim 2, it also comprises at least one dye molecule, and in wherein said dye molecule and said dsRNA and the said peptide at least one puted together.
54. composition isolated as claimed in claim 53, wherein said dye molecule are polyaromatic.
55. composition isolated as claimed in claim 53, wherein said dyestuff are fluorescent dye.
56. composition isolated according to claim 1 or claim 2, it also comprises therapeutic agent.
57. composition isolated as claimed in claim 56, wherein said therapeutic agent are cancer therapy drug.
58. composition isolated as claimed in claim 57, wherein said cancer therapy drug is selected from: paclitaxel, tamoxifen, cisplatin, amycin and vinblastine.
59. compositions as claimed in claim 58, wherein said therapeutic agent are the medicine that is used to treat metabolic disease or disease.
60. composition isolated according to claim 1 or claim 2, wherein said peptide comprises the part of the targeting moiety of toxin.
61. composition isolated as claimed in claim 60, wherein said neurotoxin are clostridial toxin.
62. composition isolated according to claim 1 or claim 2, it also comprises at least a peptide of sending.
63. composition isolated according to claim 1 or claim 2 is wherein from said first nucleotide (position 1) beginning of the said 3 ' end of said first oligonucleotide chain of said dsRNA, with the nucleotide the position of substitution 1,2 and/or 3 through modifying.
64. isolating dsRNA as claimed in claim 60, wherein said nucleotide through modification is deoxyribonucleotide.
65. isolating dsRNA according to claim 1 or claim 2, one or two in wherein said first oligonucleotide chain and said second oligonucleotide chain comprises 5 ' phosphoric acid.
66. composition isolated according to claim 1 or claim 2, at least one nucleotide of wherein said first chain or said second chain is through modifying.
67. like the described composition isolated of claim 66, wherein said through the nucleotide residue of modification be selected from 2 '-O-methyl, 2 '-methoxy ethoxy, 2 '-fluorine, 2 '-pi-allyl, 2 '-O-[2-(methylamino)-2-oxoethyl], 4 '-sulfo-, 4 '-CH2-O-2 '-bridge, 4 '-(CH2) 2-O-2 '-bridge, 2 '-LNA, 2 '-amino and 2 '-O-(N-methyl carbamate).
68. composition isolated according to claim 1 or claim 2, wherein said dsRNA in said cell by the cracking of Dicer endogenous.
69. composition isolated according to claim 1 or claim 2; Wherein in said cellular environment, the amount that is enough to reduce the said isolating double-strandednucleic acid of said expression of target gene be selected from 1 nanomole or still less, 200 picomoles or still less, 100 picomoles or still less, 50 picomoles or still less, 20 picomoles or still less or still less with 10 picomoles.
70. composition isolated according to claim 1 or claim 2, wherein said first chain and said second chain link through chemical connexon.
71. composition isolated according to claim 1 or claim 2, the said 3 ' end of wherein said first chain links through chemical connexon with the said 5 ' end of said second chain.
72. composition isolated according to claim 1 or claim 2, the nucleotide of wherein modifying with the directed process of guiding Dicer cracking replaces the nucleotide of said second chain or said first chain.
73. composition isolated according to claim 1 or claim 2; It comprises the nucleotide through modifying; Said through the nucleotide of modification be selected from deoxyribonucleotide, bi-deoxyribose nucleotide, acyclic nucleotide, 3 '-deoxyadenosine (cordycepin), 3 '-azido-3 '-AZT (AZT), 2 '; 3 '-didanosine (ddI), 2 '; 3 '-two deoxidations-3 '-sulfo-cytidine (3TC), 2 '; 3 '-two dehydrogenations-2 ', 3 '-videx (d4T), 3 '-azido-3 '-monophosphic acid nucleotide, 2 of AZT (AZT) ', 3 '-two deoxidations-3 '-sulfo-cytidine (3TC) and 2 '; 3 '-two dehydrogenations-2 ', 3 '-monophosphic acid nucleotide of videx (d4T), 4-thiouracil, 5-bromouracil, 5-iodouracil, 5-(the amino pi-allyl of 3-)-uracil, 2 '-O-alkyl ribonucleotide, 2 '-O-methyl ribonucleotides, 2 '-amino ribonucleotide, 2 '-fluorine ribonucleotide and lock nucleic acid.
74. composition isolated according to claim 1 or claim 2, it comprises the phosphoric acid backbone modification, and said phosphoric acid backbone modification is selected from phosphate ester, sulfo-phosphide and phosphotriester.
75. composition isolated according to claim 1 or claim 2, the said of the said 3 ' end of wherein said first chain is selected from deoxyribonucleotide, acyclic nucleotide and fluorescence molecule through the nucleotide residue of modifying.
76. composition isolated according to claim 1 or claim 2, it is right that at least one nucleotide of at least one nucleotide of wherein said first chain and said second chain forms base mismatch.
77. composition isolated as claimed in claim 62, the wherein said peptide of sending has the aminoacid sequence that is selected from SEQID NO:1-89.
78. composition isolated according to claim 1 or claim 2, wherein said compositions is a pharmaceutical composition.
79. a method that is used for reducing the expression of target gene of cell, it comprises:
With with compare the amount that can effectively reduce the expression of target gene in the cell with reference to dsRNA, make that desired said composition isolated contacts in cell and claim 1 or 2.
81. a method that is used for reducing the expression of target gene of animal, it comprises: with compare the amount that can effectively reduce the expression of target gene in the zooblast with reference to dsRNA, with desired said composition isolated treatment animal in claim 1 or 2.
82. like the described method of claim 81, wherein when comparing with suitable contrast dsRNA, said composition isolated has enhanced pharmacokinetics.
83. like the described method of claim 81, wherein when comparing with suitable contrast dsRNA, said dsRNA has enhanced pharmacodynamics.
84. like the described method of claim 81, wherein when comparing with suitable contrast dsRNA, said dsRNA has the toxicity of reduction.
85. like the described method of claim 81, wherein when comparing with suitable contrast dsRNA, said dsRNA has picked-up in the enhanced cell.
86. a pharmaceutical composition that is used for reducing the expression of target gene of curee's cell, it comprises and said composition isolated according to claim 1 or claim 2 and the pharmaceutically acceptable carrier of comparing the amount that can effectively reduce the expression of target gene in the cell with reference to dsRNA.
87. a synthetic method like each the desired dsRNA-peptide conjugate among the claim 1-78, it comprises through chemical method or the synthetic said dsRNA of enzyme process.
88. a test kit, it comprises like each described said dsRNA-peptide conjugate and operation instruction thereof among the claim 1-78.
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